U.S. patent number 10,808,038 [Application Number 15/348,588] was granted by the patent office on 2020-10-20 for her3/her2 bispecific antibodies binding to the beta-hairpin of her3 and domain ii of her2.
This patent grant is currently assigned to HOFFMANN-LA ROCHE INC.. The grantee listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Birgit Bossenmaier, Richard Buick, Stefan Dengl, Michael Gerg, Carmen Peess, Wolfgang Schaefer, Michael Schraeml, Claudio Sustmann.
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United States Patent |
10,808,038 |
Bossenmaier , et
al. |
October 20, 2020 |
HER3/HER2 bispecific antibodies binding to the beta-hairpin of HER3
and domain II of HER2
Abstract
The invention relates to HER3/HER2 bispecific antibodies binding
to the beta-hairpin of HER3 and domain II of HER2, their
preparation and use as medicament.
Inventors: |
Bossenmaier; Birgit (Seefeld,
DE), Buick; Richard (Belfast, GB), Dengl;
Stefan (Munich, DE), Gerg; Michael (Munich,
DE), Peess; Carmen (Tutzing, DE), Schaefer;
Wolfgang (Mannheim, DE), Schraeml; Michael
(Penzberg, DE), Sustmann; Claudio (Munich,
DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
HOFFMANN-LA ROCHE INC. (Little
Falls, NJ)
|
Family
ID: |
1000005125544 |
Appl.
No.: |
15/348,588 |
Filed: |
November 10, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170233490 A1 |
Aug 17, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/EP2015/060488 |
May 12, 2015 |
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Foreign Application Priority Data
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May 14, 2014 [EP] |
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14168323 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
47/6879 (20170801); C07K 16/32 (20130101); A61K
47/6829 (20170801); C07K 16/468 (20130101); C07K
2317/34 (20130101); A61K 2039/6068 (20130101); C07K
2317/35 (20130101); C07K 2317/77 (20130101); C07K
2317/73 (20130101); C07K 2317/92 (20130101); A61K
39/39558 (20130101); A61K 2039/505 (20130101); C07K
2317/76 (20130101); C07K 2317/31 (20130101); C07K
2317/24 (20130101); C07K 2317/94 (20130101); C07K
2317/33 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); A61K 47/68 (20170101); C07K
16/46 (20060101); C07K 16/32 (20060101); A61K
39/395 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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May 2014 |
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EP |
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WO |
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2009/074318 |
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Jun 2009 |
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WO |
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2010/059315 |
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May 2010 |
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May 2011 |
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2012/022814 |
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Feb 2012 |
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2012/125864 |
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Sep 2012 |
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WO |
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2012/150320 |
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Nov 2012 |
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2012/150321 |
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Nov 2012 |
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WO |
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2013/084148 |
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Jun 2013 |
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WO |
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WO2013134881 |
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Sep 2013 |
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WO |
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2014/067642 |
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May 2014 |
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WO |
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WO2014131019 |
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Aug 2014 |
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WO |
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2015/173248 |
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Nov 2015 |
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WO |
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Other References
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(HER-2+) cancers" Poster pp. 1 ( Dec. 10, 2012). cited by applicant
.
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with potent activity in ErbB2-overexpressing cells, positively
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Meeting, pp. 845-846 ( Apr. 20, 2010). cited by applicant .
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|
Primary Examiner: Rawlings; Stephen L
Attorney, Agent or Firm: Genentech, Inc.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/EP2015/060488 filed on
May 12, 2015, publication number WO2015/173248, which claims
priority to European Patent Application No. EP14168323.5 filed on
May 14, 2014, the disclosures of which are all incorporated herein
by reference in their entirety.
Claims
The invention claimed is:
1. An isolated bispecific antibody which binds to human HER3 and to
human HER2, wherein the antibody binds to an epitope of human HER3
within the amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
which is comprised in the polypeptide of SEQ ID NO: 18 and binds to
domain II of human HER2 comprising the amino acid sequence of SEQ
ID NO: 59.
2. The bispecific antibody according to claim 1, wherein the
bispecific antibody is bivalent.
3. An isolated nucleic acid encoding the bispecific antibody
according to claim 1.
4. A host cell comprising the nucleic acid of claim 3.
5. A method of producing a bispecific antibody according to claim
3, said method comprising culturing the host cell of claim 4 so
that the antibody is produced.
6. A pharmaceutical formulation comprising the bispecific antibody
according to claim 1 and a pharmaceutically acceptable carrier.
Description
SEQUENCE LISTING
The present application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Nov. 10,
2016, is named P32126 US_ST25.txt and is 146,013 bytes in size.
FIELD OF THE INVENTION
The invention relates to HER3/HER2 bispecific antibodies, that bind
to the beta-hairpin of HER3 and domain II of HER2, their
preparation and use as medicament.
BACKGROUND OF THE INVENTION
The HER protein family consists of 4 members: HER1, also named
epidermal growth factor receptor (EGFR) or ErbB-1, HER2, also named
ErbB-2, ErbB-3, also named HER3 and ErbB-4, also named HER4. The
ErbB family proteins are receptor tyrosine kinases and represent
important mediators of cell growth, differentiation and survival.
The HER family represent receptors proteins of different ligands
like the neuregulin (NRG) family, amphiregulin, EGF and (TGF-a).
Heregulin (also called HRG or neuregulin NRG-1) is e.g. a ligand
for HER3 and HER4.
Human HER3 (ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor
tyrosine-protein kinase erbB-3, SEQ ID NO: 3) encodes a member of
the epidermal growth factor receptor (EGFR) family of receptor
tyrosine kinases which also includes HER1 (also known as EGFR),
HER2, and HER4 (Kraus, M. H. et al, PNAS 86 (1989) 9193-9197;
Plowman, G. D. et al, PNAS 87 (1990) 4905-4909; Kraus, M. H. et al,
PNAS 90 (1993) 2900-2904). Like the prototypical epidermal growth
factor receptor, the transmembrane receptor HER3 consists of an
extracellular ligand-binding domain (ECD), a dimerization domain
within the ECD, a transmembrane domain, an intracellular protein
tyrosine kinase domain (TKD) and a C-terminal phosphorylation
domain. This membrane-bound protein has a Heregulin (HRG) binding
domain within the extracellular domain but not an active kinase
domain. It therefore can bind this ligand but not convey the signal
into the cell through protein phosphorylation. However, it does
form heterodimers with other HER family members which do have
kinase activity. Heterodimerization leads to the activation of the
receptor-mediated signaling pathway and transphosphorylation of its
intracellular domain. Dimer formation between HER family members
expands the signaling potential of HER3 and is a means not only for
signal diversification but also signal amplification. For example
the HER2/HER3 heterodimer induces one of the most important
mitogenic signals via the PI3K and AKT pathway among HER family
members (Sliwkowski M. X., et al, J. Biol. Chem. 269 (1994)
14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813-1821;
Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J.
Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L., Neoplasia 8
(2006) 613-622) For an overview of HER3 and its various
interactions within the HER receptor family and the NGR ligands
family see e.g. G Sithanandam et al Cancer Gene Therapy (2008) 15,
413-448.
Amplification of this gene and/or overexpression of its protein
have been reported in numerous cancers, including prostate,
bladder, and breast tumors. Alternate transcriptional splice
variants encoding different isoforms have been characterized. One
isoform lacks the intermembrane region and is secreted outside the
cell. This form acts to modulate the activity of the membrane-bound
form. Additional splice variants have also been reported, but they
have not been thoroughly characterized.
Interestingly in its equilibrium state, the HER3 receptor exists in
its "closed confirmation", which does mean, the heterodimerization
HER3beta-hairpin motive is tethered via non-covalent interactions
to the HER3ECD domain IV (see FIGS. 1c and 1d). It is supposed,
that the "closed" HER3 conformation can be opened via the binding
of the ligand heregulin at a specific HER3 heregulin binding site.
This takes place at the HER3 interface formed by the HER3 ECD
domains I and domain III. By this interaction it is believed, that
the HER3 receptor is activated and transferred into its "open
conformation" (see FIGS. 1e and 1b and e.g. Baselga, J. et al, Nat
Rev Cancer 9 (2009). 463-475 and Desbois-Mouthon, C., et al,
Gastroenterol Clin Biol 34 (2010) 255-259). In this open
conformation heterodimerization and transignal induction with HER2
is possible (see FIG. 1b).
WO 2003/013602 relates to inhibitors of HER activity, including HER
antibodies. WO 2007/077028 and WO 2008/100624 also relate to HER3
antibodies.
WO 97/35885 and WO2010/127181 relate to HER3 antibodies.
Human HER4 (also known as ErbB-4 ERBB4, v-erb-a erythroblastic
leukemia viral oncogene homolog 4, p180erbB4 avian erythroblastic
leukemia viral (v-erb-b2) oncogene homolog 4; SEQ ID NO:5) is a
single-pass type I transmembrane protein with multiple furin-like
cysteine rich domains, a tyrosine kinase domain, a
phosphotidylinositol-3 kinase binding site and a PDZ domain binding
motif (Plowman G D, wt al, PNAS 90:1746-50(1993); Zimonjic D B, et
al, Oncogene 10:1235-7(1995); Culouscou J M, et al, J. Biol. Chem.
268:18407-10(1993)). The protein binds to and is activated by
neuregulins-2 and -3, heparin-binding EGF-like growth factor and
betacellulin. Ligand binding induces a variety of cellular
responses including mitogenesis and differentiation. Multiple
proteolytic events allow for the release of a cytoplasmic fragment
and an extracellular fragment. Mutations in this gene have been
associated with cancer. Alternatively spliced variants which encode
different protein isoforms have been described; however, not all
variants have been fully characterized.
Anti-HER4 antibodies for use in anti-cancer therapy are known e.g.
from U.S. Pat. Nos. 5,811,098, 7,332,579 or Hollmen M, et al,
Oncogene. 28 (2009) 1309-19 (anti-ErbB-4 antibody mAb 1479).
So far it was not possible to select antibodies that specifically
bind to the beta-hairpin of HER3 (and/or HER4) as these
beta-hairpins of HER3 (or of HER4) both represent hidden epitopes,
which are not accessible in the equilibrium state of these
receptors (see FIG. 1).
Human HER2 is a transmembrane surface-bound receptor tyrosine
kinase and is normally involved in the signal transduction pathways
leading to cell growth and differentiation. HER2 is a promising
target for treatment of breast cancer as it was found to be
overexpressed in about one-quarter of breast cancer patients (Bange
et al, 2001, Nature Medicine 7:548). HER2 an oncogene and
overexpression or mutation of this receptor lead to its
constitutive activation. This drives the formation of various
cancers, like breast, oral, pancreas and lung carcinoma (Schneider
et al. 1989, Weiner et al. 1990, Hou et al. 1992, Revillion et al.
1998). HER2 is the only receptor of the HER family, which is not
expressed in the tethered conformation like HER1, HER3 and HER4
are. Instead it is expressed in an open, extended conformation on
the cell surface. In this conformation the .beta.-hairpin of
subdomain II is accessible. The antibody Pertuzumab (Perjeta.RTM.)
was shown to bind immediate to the HER2 extracellular domain (ECD)
.beta.-hairpin and surrounding region in subdomain II. The
.beta.-hairpin is essential for the formation of dimers with other
HER receptors. By binding to this epitope, Pertuzumab is able to
inhibit dimer formation and therefore the activation of subsequent
signaling cascades.
The HER2/HER3 heterodimer induces one of the most important
mitogenic signals via the PI3K and AKT pathway among HER family
members (Sliwkowski M. X., et al, J. Biol. Chem. 269 (1994)
14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813-1821;
Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J.
Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L., Neoplasia 8
(2006) 613-622). Especially the formation of HRG1.beta. induced
HER2/HER3 heterodimers plays a pivotal role in cancers with
autocrine HRG loops (Gollamudi et al. 2004). Additionally, the
results of current clinical studies indicate, that success of
anti-HER2 antibody treatments is reduced in presence of HRG1.beta.
(McDonagh et al. 2012). Pertuzumab (rhuMab 2C4, U.S. Pat. No.
7,862,817, marketed e.g as PERJETA.TM.) is a humanized monoclonal
antibody, which is designed specifically to prevent the HER2
receptor from pairing (dimerising) with other HER receptors
(EGFR/HER1, HER3 and HER4) on the surface of cells, a process that
is believed to play a role in tumor growth and survival. Pertuzumab
binds to domain II of HER2, essential for dimerization. Pertuzumab
binds specifically to the 2C4 epitope, a different epitope on the
extracellular domain of HER2 as trastuzumab. Pertuzumab is the
first in a new class of HER dimerisation inhibitors (HDIs). Through
its binding to the HER2 extracellular domain, pertuzumab blocks
ligand-activated heterodimerisation of HER2 with other HER family
members, thereby inhibiting downstream signalling pathways and
cellular processes associated with tumor growth and progression
(Franklin, M. C., et al. Cancer Cell 5 (2004) 317-328 and Friess,
T, et al. Clin Cancer Res 11 (2005) 5300-5309). Pertuzumab is a
recombinant humanized version of the murine anti-HER2 antibody 2C4
(referred to as rhuMAb 2C4 or pertuzumab) and it is described
together with the respective method of preparation in WO 01/00245
and WO 2006/007398.
SUMMARY OF THE INVENTION
The present invention relates to bispecific antibodies which bind
to the beta-hairpin of human HER3 (SEQ ID NO: 1) and domain II of
human HER2 (SEQ ID NO: 59). Both domains are responsible for the
dimerization of the respective HER receptors (homo- an/or
heterodimerization).
The invention provides the use of the beta-hairpins of HER3 (and
HER4) functionally presented in a 3-dimensional orientation within
SlyD scaffolds (see e.g FIG. 2, and the polypeptides of SEQ ID NOs.
13, and 17 to 24) to obtain HER3 antibodies or binding for use in
the generation of a bispecific HER3/HER2 antibody.
The invention provides the use of a) at least one polypeptide
selected from the group consisting of:
TABLE-US-00001 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3,
which comprises the amino acid sequence of SEQ ID NO:1; (and,
optionally b) at least one polypeptide selected from the group
consisting of:
TABLE-US-00002 SEQ ID NO: 21 TtSlyDcas-Her4, SEQ ID NO: 22
TtSlyDcys-Her4, SEQ ID NO: 23 TgSlyDser-Her4, and SEQ ID NO: 24
TgSlyDcys-Her4,)
in a method for selecting an antibody, in particular an antibody
that binds to human HER3 (and binds to human HER4) for use in the
generation of a bispecific HER3/HER2 antibody, wherein the
antibody, binds within an amino acid sequence of
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3; and such HER3
antibody is then used to generate a bispecific HER3/HER2
antibody.
The invention provides a bispecific antibody which binds to human
HER3 and to human HER2, wherein the antibody binds within an amino
acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is
comprised in a polypeptide selected from the group consisting
of:
TABLE-US-00003 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3.
The invention provides a bispecific antibody which binds to human
HER3 and to human HER2, wherein the antibody binds within an amino
acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is
comprised in a polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3).
One embodiment of the invention is a bispecific antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to domain II of human HER2 (SEQ ID NO: 59). One
embodiment of the invention is a bispecific antibody which antibody
binds to the polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) which
comprises the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and which antibody binds to domain II of human HER2 n(SEQ ID NO:
59).
One embodiment of the invention is a bispecific antibodies that
binds to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) and that binds to the same epitope on human HER2 as
pertuzumab. One embodiment of the invention is a bispecific
antibody which antibody binds to the polypeptide of SEQ ID NO: 18
(TtSlyDcys-Her3) which comprises the amino acid sequence
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and which antibody binds to the
same epitope on human HER2 as pertuzumab.
One embodiment of the invention is a bispecific antibodies that
binds to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) and that competes for binding to human HER2 with pertuzumab.
One embodiment of the invention is a bispecific antibody which
antibody binds to the polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3)
which comprises the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) and which antibody competes for binding to human HER2 with
pertuzumab.
One embodiment of the invention is a bispecific antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to human HER2 and comprises all six heavy and light
chains HVRs of pertuzumab (SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:
62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65). One
embodiment of the invention is a bispecific antibodies that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to human HER2 and comprises the VH and VL of
pertuzumab (SEQ ID NO: 66 and SEQ ID NO. 67)).
One embodiment is a multispecific antibody that binds to human HER3
and human HER2 as described above which binds also to human HER4.
In one embodiment such multispecific antibody that binds to human
HER3 and human HER2 binds also to the beta-hairpin of human HER4
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2). In one embodiment such
multispecific antibody that binds to human HER3 and human HER2 also
binds to the polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) which
comprises the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID
NO:2).
In one embodiment such bispecific HER3/HER2 does not crossreact
with human HER4. In one embodiment such bispecific HER3/HER2 does
not crossreact with the beta-hairpin of human HER4
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2). In one embodiment such
bispecific HER3/HER2 does not crossreact with the polypeptide of
SEQ ID NO: 22 (TtSlyDcys-Her4) which comprises the amino acid
sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).
One embodiment of the invention is a bispecific antibody
a) that binds to human HER3 and comprises the heavy chain HVRs
of
SEQ ID NO: 25 heavy chain HVR-H1, M-05-74,
SEQ ID NO: 26 heavy chain HVR-H2, M-05-74, and
SEQ ID NO: 27 heavy chain HVR-H3, M-05-74,
and comprises the light chain heavy chain HVRs of
SEQ ID NO: 28 light chain HVR-L1, M-05-74,
SEQ ID NO: 29 light chain HVR-L2, M-05-74, and
SEQ ID NO: 30 light chain HVR-L3, M-05-74;
and
b) that binds to human HER2 and comprises the heavy chain HVRs
of
SEQ ID NO: 60 heavy chain HVR-H1, pertuzumab,
SEQ ID NO: 61 heavy chain HVR-H2 pertuzumab,
SEQ ID NO: 62 heavy chain HVR-H3, pertuzumab,
and comprises the light chain heavy chain HVRs of
SEQ ID NO: 63 light chain HVR-L1, pertuzumab,
SEQ ID NO: 64 light chain HVR-L2, pertuzumab, and
SEQ ID NO: 65 light chain HVR-L3 pertuzumab.
One embodiment of the invention is a bispecific antibody
a) that binds to human HER3 and comprises
i) a variable heavy chain domain VH with the amino acid sequence of
SEQ ID NO:33 and a variable light chain domain VL with the amino
acid sequence of SEQ ID NO:41,
ii) a variable heavy chain domain VH with the amino acid sequence
of SEQ ID NO:33 and a variable light chain domain VL with the amino
acid sequence of SEQ ID NO:39, or
iii) a variable heavy chain domain VH with the amino acid sequence
of SEQ ID NO:33 and a variable light chain domain VL with the amino
acid sequence of SEQ ID NO:42;
and
b) that binds to human HER2 and a variable heavy chain domain VH
with the amino acid sequence of SEQ ID NO:66 and a variable light
chain domain VL with the amino acid sequence of SEQ ID NO:67.
In one preferred embodiment such bispecific antibody is
bivalent.
The invention further provides an isolated nucleic acid encoding
such bispecific HER3/HER2 antibody.
The invention further provides a host cell comprising such nucleic
acid.
The invention further provides a method of producing such antibody
comprising culturing such host cell so that the antibody is
produced.
In on embodiment such method further comprises recovering such
antibody from the host cell.
The invention further provides an immunoconjugate comprising such
bispecific HER3/HER2 and a cytotoxic agent.
The invention further provides a pharmaceutical formulation
comprising such bispecific HER3/HER2 antibody and a
pharmaceutically acceptable carrier.
The invention further provides the bispecific HER3/HER2 antibody
described herein for use as a medicament. The invention further
provides the bispecific HER3/HER2 antibody described herein, or the
immunoconjugate comprising the bispecific HER3/HER2 antibody and a
cytotoxic agent, for use in treating cancer. The invention further
provides the bispecific HER3/HER2 antibody described herein for use
in inhibition of HER3/HER2 dimerization and/or HER2/HER2
dimerization.
Use of such bispecific HER3/HER2 antibody, or an immunoconjugate
comprising the bispecific HER3/HER2 antibody and a cytotoxic agent,
in the manufacture of a medicament. Such use wherein the medicament
is for treatment of cancer. Such use wherein the medicament is for
the inhibition of HER3/HER2 dimerization and/or HER2/HER2
dimerization.
The invention further provides a method of treating an individual
having cancer comprising administering to the individual an
effective amount of the bispecific HER3/HER2 antibody described
herein, or an immunoconjugate comprising the bispecific HER3/HER2
antibody and a cytotoxic agent.
The invention further provides a method of inhibiting growth of a
tumor cell in an individual suffering from cancer comprising
administering to the individual an effective amount of the
bispecific HER3/HER2 antibody as described herein, thereby
inhibiting growth of a tumor cell in the individual.
Disclosed is a polypeptide selected from the group consisting
of:
TABLE-US-00004 i) SEQ ID NO: 13 TtSlyD-FKBP-Her3, ii) SEQ ID NO: 17
TtSlyDcas-Her3, iii) SEQ ID NO: 18 TtSlyDcys-Her3, iv) SEQ ID NO:
19 TgSlyDser-Her3, and v) SEQ ID NO: 20 TgSlyDcys-Her3,
which polypeptide comprises the amino acid sequence of SEQ ID
NO:1
Disclosed is a polypeptide selected from the group consisting
of:
TABLE-US-00005 i) SEQ ID NO: 21 TtSlyDcas-Her4, ii) SEQ ID NO: 22
TtSlyDcys-Her4, iii) SEQ ID NO: 23 TgSlyDser-Her4, and iv) SEQ ID
NO: 24 TgSlyDcys-Her4,
which polypeptide comprises the amino acid sequence of SEQ ID
NO:2.
Using the beta-hairpins of HER3 (and HER4) functionally presented
in a 3-dimensional orientation within SlyD scaffolds (see e.g FIG.
2, and the polypeptides of SEQ ID NOs. 13, and 17 to 24) the
bispecific HER3/HER2 antibodies, described herein binding to these
beta-hairpins could be selected.
It was found that the antibodies, according to the invention can
have highly valuable properties such as strong growth inhibition of
HER3 expressing cancer cells, strong inhibition of HER3 mediated
signal transduction (such as e.g. HER3 phosphorylation) which is
related to cancer cell proliferation, or very specific
pharmacokinetic properties (such as faster association rates and
higher Molar Ratios of the binding the activated HER3 in the
presence of Heregulin ("open conformation) when compared to the
absence of Heregulin ("closed conformation"). Furthermore they show
strong tumor growth inhibition and are able to efficiently inhibit
HER3/HER2 dimerization and/or HER2/HER2 dimerization.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-E Schematic overview of "closed" and "open" HER3
conformation and the influence of the Neuregulin family ligands
(like e.g. Heregulin abbreviated here as HR) on the conformation
change.
FIG. 2 3D-structure of the beta-hairpin of HER3 functionally
presented in a 3-dimensional orientation within a SlyD scaffold of
Thermus thermophiles.
FIG. 3 SDS-PAGE analysis of Ni-NTA purification of
TtSlyD-FKBP-Her3. E1 and E2 show the purified fractions 12 and
13.SN: E. coli lysate supernatant before purification.
FIG. 4 SEC elution profile of a Ni-NTA purified fraction of Thermus
thermophilus SlyD-FKBP-Her-3.
FIG. 5 Testing of specificity and reactivity in IHC of the selected
clones. All three clones showed binding to Her3 and cross
reactivity against Her4. No cross reactivity against Her1 and Her2
was detectable.
FIG. 6 FACS analysis of M-05-74 antibody induced time dependent
HER3 internalization in T47D cells.
FIG. 7 Biacore.TM. sensorgram overlay plot. 1: 100 nM
M-05-74*Heregulin/Her-3 ECD interaction. 2: 100 nM
M-08-11*Heregulin/Her-3 ECD interaction. 3&4: 100 nM M-05-74
and 100 nM M-08-11*Her-3 ECD interaction. 5: buffer reference.
FIG. 8 Sensorgram overlay of the Biacore.TM. epitope-binning
experiment. The primary antibody M-05-74 (M-074 in the Figure)
presented the Her-3 ECD to the secondary antibodies M-208, GT
(=8B8), M-05-74 and M-08-11 (M-011 in the FIG. 8) (M-. The noise of
the measurement was 5 RU.
FIG. 9 Biacore.TM. sensorgram overlay plot. 1: 90 nM
Heregulin*Her-3 ECD complex on M-05-74. 2: 90 nM Heregulin*Her-3
ECD complex on M-08-11. 3: 90 nM Heregulin*Her-3 ECD complex on 8B8
antibody.
FIG. 10 Schematic Mode of Actions identified by Biacore.TM.
functional assays. 1: M-08-11 binds to the Heregulin activated
Her-3 ECD and induces a delayed Heregulin dissociation, whereby
M-08-11 stays in the Her-3 ECD receptor complex. 2: M-05-74 binds
to the Heregulin activated Her-3 ECD. Heregulin is trapped in the
complex and the antibody stays in the complex 3: 8B8 binds the
Heregulin activated Her-3 ECD. The whole complex dissociates from
the antibody.
FIG. 11 Strategy of the epitope mapping and alanine-scan approach.
The peptide hairpin sequences (peptide hairpin) of EGFR (amino
acids 232-271 of SEQ ID NO: 7), Her-2 ECD (amino acids 239-277 of
SEQ ID NO: 9), Her-3 ECD (amino acids 232-271 of SEQ ID NO: 4), and
Her-4 ECD (amino acids 229-268 of SEQ ID NO: 6), including their
structural embeddings (structural) were investigated. Cysteins were
replaced by serines.
FIG. 12 CelluSpots.TM. Synthesis and Epitope Mapping of epitopes of
antibody M-05-74 on HER3 and HER4. Anti-HER3/HER4 antibody M-05-74
binds to HER3 ECD binding epitope VYNKLTFQLEP (SEQ ID NO:43) and to
HER4 ECD binding epitope VYNPTTFQLE (SEQ ID NO:44).
FIG. 13 Results from the CelluSpots.TM. Ala-Scan of anti HER3/HER4
antibody M-05-74 (named M-074 in the Figure) and anti-HER3 antibody
M-08-11 (named M-011) with no HER4 crossreactivity)--the amino
acids which are contributing most to the binding of anti-HER3/HER4
antibody M-05-74 to its HER3 ECD binding epitope VYNKLTFQLEP (SEQ
ID NO:43) and to its HER4 ECD binding epitope VYNPTTFQLE (SEQ ID
NO:44) are underlined/bold.
FIG. 14 Binding of M-05-74 (M-074) induces/promotes binding of HRG
to the HER3-ECD.
FIG. 15 Inhibition of HER2/HER3 heterodimers/heterodimerization
(Imunoprecipitation and Western Blot) in MCF7 cells
(HER3-IP=immunoprecipitation with HER3
antibody/HER2-IP=immunoprecipitation with HER3 antibody).
FIG. 16 Treatment of MDA-MB175 cells with M-05-74 resulted in
inhibition of cell proliferation.
FIG. 17 Treatment with M-05-74 (M-074) (10 mg/kg q7d, i.p.)
resulted in tumor stasis a FaDu HNSCC transplanted xenografts.
FIG. 18 Treatment with M-05-74-Fab-Pseudomonas exotoxin conjugate
(M-074-PE) (10 mg/kg q7d, i.p.) resulted in stronger inhibition of
cell proliferation in the presence (bold line) of HRG than in the
absence (thin line) of HRG.
FIG. 19 In vivo tumor cell growth inhibition by
M-05-74-Fab-Pseudomonas exotoxin conjugate (M-05-74-PE). Legend:
closed line (vehicle); dotted line (M-05-74-Fab-Pseudomonas
exotoxin conjugate (M-05-74-PE)).
FIG. 20 Biacore.TM. sensorgram overlay plot: binding of the
antibody M-05-74 (1) of the present invention to TtSlyDcys-Her3
(SEQ ID NO: 18) in comparison with anti-HER3 antibody MOR09823 (2)
described in WO2012/22814. While the antibody of the present
M-05-74 (1) shows a clear binding signal to TtSlyDcys-Her3 (SEQ ID
NO: 18), the antibody anti-HER3 antibody MOR09823 (2) shows no
binding at all to TtSlyDcys-Her3 (SEQ ID NO: 18). Control
measurement (3) without antibody at all did not show any binding to
TtSlyDcys-Her3 (SEQ ID NO: 18).
FIG. 21 Selection of optimized humanized M-05-74 antibody via
ribosome display: Analytical DNA chip electrophorese of PCR
products obtained after reverse transcription of the enriched RNA
during display selection. The obtained gel image shows enrichment
of selected construct DNA in lane 1 and no enrichment for the
negative control--panning without antigen--in lane 2. The remaining
controls are also negative as expected. The DNA digest was complete
(lane 3 for target, lane 4 for background). Therefore all obtained
DNA in lane 1 is derived from binding variants, selected in the
panning step, and their corresponding RNA. Neither the negative
control of the reverse transcription, nor the negative control of
the PCR is showing bands. Lane 7 shows the product of the pooled
PCR reactions after purification.
FIG. 22 Expression vector construct-DIB light chain (VL-CK).
FIG. 23 Expression vector construct-DIB heavy chain with `knob`
amino acid in CH3 (VH-CH1-CH2-CH3(knob)).
FIG. 24 Expression vector construct-Pertuzumab crossed light chain
(VL-CH1).
FIG. 25 Expression vector construct-Pertuzumab crossed heavy chain
with `hole` mutation in CH3 (VH-CK-CH2-CH3(hole)).
FIGS. 26A-B (A) Layout of the bispecific CrossMab DIB.times.PERT as
a hybrid of DIB-74 and Pertuzumab: The Scheme shows the bispecific
CrossMab DIB.times.PERT and its parental antibodies DIB-74 and
Pertuzumab. DIB-74 and Pertuzumab bind to the .beta.-hairpins of
the HER3-ECD and the HER2-ECD, respectively. Dark colors indicate
Ig heavy chains, light colors Ig light chains. The CH3 Ig domains
contain `knob` or `hole` mutations, according to the
`knob-into-hole` technology. A domain cross-over of CH1 and CK of
the Pertuzumab heavy and light chain was designed to facilitate the
correct light chain-heavy chain assimilation. (B) Scheme of
DIB-MoAb, an artificial monovalent antibody format as a derivative
of DIB-74. The `knob-into-hole` technology and a CH1-CK domain
cross-over were applied.
FIGS. 27A-C Qualitative analytic of the purified DIB.times.PERT
CrossMab by GPC and SDS-PAGE: The DIB.times.PERT end product
quality was assessed, using GPC and SDS-PAGE. (A) Analytic GPC
peaks were numbered consecutively (1-7). (B) Tabular presentation
of all seven GF30 peaks, listing retention times, absorption at 280
nm and the relative peak area in percentage. (C) The Coomassie
staining of a 4-12% SDS-PAGE showing the DIB.times.PERT end product
under reducing (+) and non-reducing (-) conditions. DIB.times.PERT
and DIB.times.PERT heavy and light chains are indicated by
arrows.
FIG. 28 Comparison of kinetic characteristics of DIB.times.PERT and
the parental antibodies by SPR: Antibodies were captured on a CM5
sensor chip surface and kinetic interactions at 25.degree. C. with
soluble analytes were measured, using a Biacore.TM. B3000
instrument (GE Healthcare, Munchen, Germany). Analytes were
injected for 5 minutes and dissociation was recorded for 10
minutes. The analytes HER2-ECD and HER3-ECD/HRG1.beta. were
injected in a five-step 1:3 series dilution with a highest
concentration of 270 nM.
FIGS. 29A-D Simultaneous complex-formation of HER2-ECD and
activated HER3-ECD by DIB.times.PERT in solution: Antibodies were
captured on a CM5 sensor chip surface and kinetic interactions at
25.degree. C. with soluble analytes were measured, using a
Biacore.TM. B3000 instrument (GE Healthcare, Munchen, Germany).
Analytes HER2-ECD and HER3-ECD/HRG1.beta. were sequentially
injected for 8 minutes and dissociation was recorded for 5 minutes.
(A and C) Assay setup for sensorgrams (B) and (D), respectively. (B
and D) The sensorgrams show the sequential binding of both
analytes. Analyze injections and the molar ratio are indicated with
arrows and `MR`, respectively.
FIGS. 30A-B Growth proliferation inhibition of MDA-MB-175 VII
cancer cells by DIB.times.PERT in comparison to parental
antibodies. MDA-MB-175 VII breast cancer cells were incubated for 6
days with a series dilution of either of the following antibodies:
DIB.times.PERT, DIB-MoAb, DIB-74, Pertuzumab (PERT), RG7116, DIB-74
and Pertuzumab, RG7116 and Pertuzumab and an Isotype control.
EC.sub.50 values were calculated using means of triplicates for
each antibody concentration. Depicted are normalized four-parameter
sigmoidal dose-response curves. Standard deviations are indicated
as error bars.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
An "affinity matured" antibody refers to an antibody with one or
more alterations in one or more hypervariable regions (HVRs),
compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
The terms "bispecific HER3/HER2 antibody", "a bispecific
(HER3/HER2) antibody that binds to (human) HER3 and that binds to
(human) HER2" and "a bispecific (HER3/HER2) antibody that
specifically binds to (human) HER3 and that specifically binds to
(human) HER2" refer to an antibody that is capable of binding HER3
with sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting HER3 and is
capable of binding HER2 with sufficient affinity such that the
antibody is useful as a diagnostic and/or therapeutic agent in
targeting HER2. In one embodiment, the extent of binding of an
bispecific HER3/HER2 antibody to an unrelated, non-HER3 protein
(except of HER4) is less than about 10% of the binding of the
antibody to HER3 or HER2 as measured, e.g., by a Surface Plasmon
Resonance assay (e.g. BIACORE). In certain embodiments, an antibody
that binds to human HER3 or HER2 has a KD value of the binding
affinity for binding to human HER3 or HER2 of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). In certain embodiments the antibody according to the
invention, binds (also) to human HER4 and has a KD value of the
binding affinity for binding to human HER4 of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M). In one preferred embodiment the respective KD value
of the binding affinities is determined in a Surface Plasmon
Resonance assay using the wildtype Extracellular domain (ECD) of
human HER3 or HER2 (HER3-ECD or HER2-ECD) for the HER3 binding
affinity or HER2 binding affinity, respectively. and wildtype human
HER4-ECD for the HER4 binding affinity, respectively. In case the
bispecific HER3/HER2 antibody also binds to (human) HER4, the terms
"bispecific HER3/HER2 antibody", "an bispecific (HER3/HER2)
antibody that binds to (human) HER3 and that binds to (human) HER2"
and "an bispecific (HER3/HER2) antibody that specifically binds to
(human) HER3 and that specifically binds to (human) HER2" refer to
a "multispecific HER3/HER2 antibody that also binds to (human)
HER4", "a multispecific (HER3/HER2) antibody that binds to (human)
HER3 and that binds to (human) HER2 that also binds to (human)
HER4" and "a multispecific (HER3/HER2) antibody that specifically
binds to (human) HER3 and that specifically binds to (human) HER2
that also binds to (human) HER4".
The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
An "antibody fragment" refers to a molecule other than an intact
antibody that comprises a portion of an intact antibody that binds
the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific.
"Bispecific antibodies" according to the invention are antibodies
which have two different antigen-binding specificities. Antibodies
of the present invention are specific for two different antigens,
VEGF as first antigen and ANG-2 as second antigen.
The term "monospecific" antibody as used herein denotes an antibody
that has one or more binding sites each of which bind to the same
epitope of the same antigen.
The term "valent" as used within the current application denotes
the presence of a specified number of binding sites in an antibody
molecule. As such, the terms "bivalent", "tetravalent", and
"hexavalent" denote the presence of two binding site, four binding
sites, and six binding sites, respectively, in an antibody
molecule. In one preferred embodiment of the invention the
bispecific antibodies according to the invention are
"bivalent".
The term "cancer" as used herein may be, for example, lung cancer,
non small cell lung (NSCL) cancer, bronchioloalviolar cell lung
cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the
head or neck, cutaneous or intraocular melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, gastric cancer, colon cancer, breast cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma,
lymphocytic leukemia, including refractory versions of any of the
above cancers, or a combination of one or more of the above
cancers. In one preferred embodiment such cancer is a breast
cancer, ovarian cancer, cervical cancer, lung cancer or prostate
cancer. In one preferred embodiment such cancers are further
characterized by HER3 expression (or overexpression). In one
preferred embodiment such cancers are additionally further
characterized by HER2 expression (or overexpression). One further
embodiment the invention are the bispecific HER3/HER2 antibodies of
the present invention for use in the simultaneous treatment of
primary tumors and new metastases.
The term "chimeric" antibody refers to an antibody in which a
portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
The "class" of an antibody refers to the type of constant domain or
constant region possessed by its heavy chain. There are five major
classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into subclasses (isotypes), e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu. respectively.
The term "cytotoxic agent" as used herein refers to a substance
that inhibits or prevents a cellular function and/or causes cell
death or destruction. Cytotoxic agents include, but are not limited
to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186,
Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof and the various antitumor or anticancer
agents disclosed below. In one preferred embodiment the "cytotoxic
agent" is Pseudomonas exotoxin A or variants thereof. In one
preferred embodiment the "cytotoxic agent" is amatoxin or a
variants thereof.
"Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
The term "epitope" includes any polypeptide determinant capable of
specific binding to an antibody. In certain embodiments, epitope
determinant include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody.
The term "Fc region" herein is used to define a C-terminal region
of an immunoglobulin heavy chain that contains at least a portion
of the constant region.
The term includes native sequence Fc regions and variant Fc
regions. In one embodiment, a human IgG heavy chain Fc region
extends from Cys226, or from Pro230, to the carboxyl-terminus of
the heavy chain. However, the C-terminal lysine (Lys447) of the Fc
region may or may not be present. Unless otherwise specified
herein, numbering of amino acid residues in the Fc region or
constant region is according to the EU numbering system, also
called the EU index, as described in Kabat, E. A. et al., Sequences
of Proteins of Immunological Interest, 5th ed., Public Health
Service, National Institutes of Health, Bethesda, Md. (1991), NIH
Publication 91-3242.
"Framework" or "FR" refers to variable domain residues other than
hypervariable region (HVR) residues. The FR of a variable domain
generally consists of four FR domains: FR1, FR2, FR3, and FR4.
Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
The terms "full length antibody," "intact antibody," and "whole
antibody" are used herein interchangeably to refer to an antibody
having a structure substantially similar to a native antibody
structure or having heavy chains that contain an Fc region as
defined herein.
The terms "host cell," "host cell line," and "host cell culture"
are used interchangeably and refer to cells into which exogenous
nucleic acid has been introduced, including the progeny of such
cells. Host cells include "transformants" and "transformed cells,"
which include the primary transformed cell and progeny derived
therefrom without regard to the number of passages. Progeny may not
be completely identical in nucleic acid content to a parent cell,
but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
A "human antibody" is one which possesses an amino acid sequence
which corresponds to that of an antibody produced by a human or a
human cell or derived from a non-human source that utilizes human
antibody repertoires or other human antibody-encoding sequences.
This definition of a human antibody specifically excludes a
humanized antibody comprising non-human antigen-binding
residues.
A "human consensus framework" is a framework which represents the
most commonly occurring amino acid residues in a selection of human
immunoglobulin VL or VH framework sequences. Generally, the
selection of human immunoglobulin VL or VH sequences is from a
subgroup of variable domain sequences. Generally, the subgroup of
sequences is a subgroup as in Kabat, E. A. et al., Sequences of
Proteins of Immunological Interest, 5th ed., Bethesda Md. (1991),
NIH Publication 91-3242, Vols. 1-3. In one embodiment, for the VL,
the subgroup is subgroup kappa I as in Kabat et al., supra. In one
embodiment, for the VH, the subgroup is subgroup III as in Kabat et
al., supra.
A "humanized" antibody refers to a chimeric antibody comprising
amino acid residues from non-human HVRs and amino acid residues
from human FRs. In certain embodiments, a humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the HVRs
(e.g., CDRs) correspond to those of a non-human antibody, and all
or substantially all of the FRs correspond to those of a human
antibody. A humanized antibody optionally may comprise at least a
portion of an antibody constant region derived from a human
antibody. A "humanized variant" of an antibody, e.g., a non-human
antibody, refers to an antibody that has undergone humanization. In
one preferred embodiment, a murine HVR is grafted into the
framework region of a human antibody to prepare the "humanized
antibody." See e.g. Riechmann, L., et al., Nature 332 (1988)
323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270.
The murine variable region amino acid sequence is aligned to a
collection of human germline antibody V-genes, and sorted according
to sequence identity and homology. The acceptor sequence is
selected based on high overall sequence homology and optionally
also the presence of the right canonical residues already in the
acceptor sequence (see Poul, M-A. and Lefranc, M-P., in "Ingenierie
des anticorps banques combinatores" ed. by Lefranc, M-P. and
Lefranc, G., Les Editions INSERM, 1997). The germline V-gene
encodes only the region up to the beginning of HVR3 for the heavy
chain, and till the middle of HVR3 of the light chain. Therefore,
the genes of the germline V-genes are not aligned over the whole
V-domain. The humanized construct comprises the human frameworks 1
to 3, the murine HVRs, and the human framework 4 sequence derived
from the human JK4, and the JH4 sequences for light and heavy
chain, respectively. Before selecting one particular acceptor
sequence, the so-called canonical loop structures of the donor
antibody can be determined (see Morea, V., et al., Methods, Vol 20,
Issue 3 (2000) 267-279). These canonical loop structures are
determined by the type of residues present at the so-called
canonical positions. These positions lie (partially) outside of the
HVR regions, and should be kept functionally equivalent in the
final construct in order to retain the HVR conformation of the
parental (donor) antibody.
The term "hypervariable region" or "HVR" as used herein refers to
each of the regions of an antibody variable domain which are
hypervariable in sequence ("complementarity determining regions" or
"CDRs") and/or form structurally defined loops ("hypervariable
loops") and/or contain the antigen-contacting residues ("antigen
contacts"). Generally, antibodies comprise six HVRs: three in the
VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs
herein include:
(a) hypervariable loops occurring at amino acid residues 26-32
(L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101
(H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2),
89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991));
(c) antigen contacts occurring at amino acid residues 27c-36 (L1),
46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3)
(MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) combinations of (a), (b), and/or (c), including HVR amino acid
residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35
(H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
An "immunoconjugate" is an antibody conjugated to one or more
heterologous molecule(s), including but not limited to a cytotoxic
agent.
An "individual" or "subject" is a mammal. Mammals include, but are
not limited to, domesticated animals (e.g., cows, sheep, cats,
dogs, and horses), primates (e.g., humans and non-human primates
such as monkeys), rabbits, and rodents (e.g., mice and rats). In
certain embodiments, the individual or subject is a human.
An "isolated" antibody is one which has been separated from a
component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.
An "isolated" nucleic acid refers to a nucleic acid molecule that
has been separated from a component of its natural environment. An
isolated nucleic acid includes a nucleic acid molecule contained in
cells that ordinarily contain the nucleic acid molecule, but the
nucleic acid molecule is present extrachromosomally or at a
chromosomal location that is different from its natural chromosomal
location.
"Isolated nucleic acid encoding an bispecific HER3/HER2 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical and/or bind the same epitope, except for possible variant
antibodies, e.g., containing naturally occurring mutations or
arising during production of a monoclonal antibody preparation,
such variants generally being present in minor amounts. In contrast
to polyclonal antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody of a monoclonal antibody
preparation is directed against a single determinant on an antigen.
Thus, the modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
The term "Mab" refers to monoclonal antibodies, whereas the term
"hMab" refers to humanized variants of such monoclonal
antibodies.
A "naked antibody" refers to an antibody that is not conjugated to
a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The
naked antibody may be present in a pharmaceutical formulation.
(Include if Prior art has immunoconjugates).
"Native antibodies" refer to naturally occurring immunoglobulin
molecules with varying structures. For example, native IgG
antibodies are heterotetrameric glycoproteins of about 150,000
daltons, composed of two identical light chains and two identical
heavy chains that are disulfide-bonded. From N- to C-terminus, each
heavy chain has a variable region (VH), also called a variable
heavy domain or a heavy chain variable domain, followed by three
constant domains (CH1, CH2, and CH3). Similarly, from N- to
C-terminus, each light chain has a variable region (VL), also
called a variable light domain or a light chain variable domain,
followed by a constant light (CL) domain. The light chain of an
antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
"Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino
acid sequence A to, with, or against a given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with, or against a given amino acid sequence B) is calculated
as follows: 100 times the fraction X/Y where X is the number of
amino acid residues scored as identical matches by the sequence
alignment program ALIGN-2 in that program's alignment of A and B,
and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence A
is not equal to the length of amino acid sequence B, the % amino
acid sequence identity of A to B will not equal the % amino acid
sequence identity of B to A. Unless specifically stated otherwise,
all % amino acid sequence identity values used herein are obtained
as described in the immediately preceding paragraph using the
ALIGN-2 computer program.
The term "pharmaceutical formulation" refers to a preparation which
is in such form as to permit the biological activity of an active
ingredient contained therein to be effective, and which contains no
additional components which are unacceptably toxic to a subject to
which the formulation would be administered.
A "pharmaceutically acceptable carrier" refers to an ingredient in
a pharmaceutical formulation, other than an active ingredient,
which is nontoxic to a subject. A pharmaceutically acceptable
carrier includes, but is not limited to, a buffer, excipient,
stabilizer, or preservative.
The term "HER3," as used herein, refers to any native HER3 from any
vertebrate source, including mammals such as primates (e.g. humans)
and rodents (e.g., mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed HER3 as well as any
form of HER3 that results from processing in the cell. The term
also encompasses naturally occurring variants of HER3, e.g., splice
variants or allelic variants. The amino acid sequence of an
exemplary human HER3 is shown in SEQ ID NO:3. "Human HER3" (ErbB-3,
ERBB3, c-erbB-3,c-erbB3, receptor tyrosine-protein kinase erbB-3,
SEQ ID NO: 3) encodes a member of the epidermal growth factor
receptor (EGFR) family of receptor tyrosine kinases which also
includes HER1 (also known as EGFR), HER2, and HER4 (Kraus, M. H. et
al, PNAS 86 (1989) 9193-9197; Plowman, G. D. et al, PNAS 87 (1990)
4905-4909; Kraus, M. H. et al, PNAS 90 (1993) 2900-2904). Like the
prototypical epidermal growth factor receptor, the transmembrane
receptor HER3 consists of an extracellular ligand-binding domain
(ECD), a dimerization domain within the ECD, a transmembrane
domain, an intracellular protein tyrosine kinase domain (TKD) and a
C-terminal phosphorylation domain. This membrane-bound protein has
a Heregulin (HRG) binding domain within the extracellular domain
but not an active kinase domain. It therefore can bind this ligand
but not convey the signal into the cell through protein
phosphorylation. However, it does form heterodimers with other HER
family members which do have kinase activity. Heterodimerization
leads to the activation of the receptor-mediated signaling pathway
and transphosphorylation of its intracellular domain. Dimer
formation between HER family members expands the signaling
potential of HER3 and is a means not only for signal
diversification but also signal amplification. For example the
HER2/HER3 heterodimer induces one of the most important mitogenic
signals via the PI3K and AKT pathway among HER family members
(Sliwkowski M. X., et al, J. Biol. Chem. 269 (1994) 14661-14665;
Alimandi M, et al, Oncogene. 10 (1995) 1813-1821; Hellyer, N.J., J.
Biol. Chem. 276 (2001) 42153-4261; Singer, E., J. Biol. Chem. 276
(2001) 44266-44274; Schaefer, K. L., Neoplasia 8 (2006) 613-622)
For an overview of HER3 and its various interactions within the HER
receptor family and the NGR ligands family see e.g. G Sithanandam
et al Cancer Gene Therapy (2008) 15, 413-448.
Interestingly in its equilibrium state, the HER3 receptors exists
in its "closed confirmation", which does mean, the
heterodimerization HER3 beta-hairpin motive is tethered via
non-covalent interactions to the HER3 ECD domain IV (see FIG. 1c).
It is supposed, that the "closed" HER3 conformation can be opened
via the binding of the ligand heregulin at a specific HER3
heregulin binding site. This takes place at the HER3 interface
formed by the HER3 ECD domains I and domain III. By this
interaction it is believed, that the HER3 receptor is activated and
transferred into its "open conformation" (see FIG. 1b and e.g.
Baselga, J. et al, Nat Rev Cancer 9 (2009). 463-475 and
Desbois-Mouthon, C., at al, Gastroenterol Clin Biol 34 (2010)
255-259). In this open conformation heterodimerization and
transignal induction with HER2 is possible (see FIG. 1b).
The term "HER2," as used herein, refers to any native HER2 from any
vertebrate source, including mammals such as primates (e.g. humans)
and rodents (e.g., mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed HER2 as well as any
form of HER2 that results from processing in the cell. The term
also encompasses naturally occurring variants of HER4, e.g., splice
variants or allelic variants. The amino acid sequence of an
exemplary human HER2 is shown in SEQ ID NO:5. "Human HER2" (also
known as c-erb B2/neu protein, p185erbB2, proto-oncogene Neu,
proto-oncogene c-ErbB-2, receptor tyrosine-protein kinase erbB-2,
v-erb-b2 erythroblastic leukemia viral oncogene homolog 2,
neuro/glioblastoma derived oncogene homolog; SEQ ID NO:9) is a
transmembrane surface-bound receptor tyrosine kinase and is
normally involved in the signal transduction pathways leading to
cell growth and differentiation. HER2 is a promising target for
treatment of breast cancer as it was found to be overexpressed in
about one-quarter of breast cancer patients (Bange et al, 2001,
Nature Medicine 7:548). HER2 an oncogene and overexpression or
mutation of this receptor lead to its constitutive activation. This
drives the formation of various cancers, like breast, oral,
pancreas and lung carcinoma (Schneider et al. 1989, Weiner et al.
1990, Hou et al. 1992, Revillion et al. 1998). HER2 is the only
receptor of the HER family, which is not expressed in the tethered
conformation like HER1, HER3 and HER4 are. Instead it is expressed
in an open, extended conformation on the cell surface. In this
conformation the .beta.-hairpin of subdomain II is accessible. The
antibody Pertuzumab (Perjeta.RTM.) was shown to bind immediate to
the HER2 extracellular domain (ECD) .beta.-hairpin and surrounding
region in subdomain II. The .beta.-hairpin is essential for the
formation of dimers with other HER receptors. By binding to this
epitope, Pertuzumab is able to inhibit dimer formation and
therefore the activation of subsequent signaling cascades.
The HER2/HER3 heterodimer induces one of the most important
mitogenic signals via the PI3K and AKT pathway among HER family
members (Sliwkowski M. X., et al, J. Biol. Chem. 269 (1994)
14661-14665; Alimandi M, et al, Oncogene. 10 (1995) 1813-1821;
Hellyer, N.J., J. Biol. Chem. 276 (2001) 42153-4261; Singer, E., J.
Biol. Chem. 276 (2001) 44266-44274; Schaefer, K. L., Neoplasia 8
(2006) 613-622). Especially the formation of HRG1.beta. induced
HER2/HER3 heterodimers plays a pivotal role in cancers with
autocrine HRG loops (Gollamudi et al. 2004). Additionally, the
results of current clinical studies indicate, that success of
anti-HER2 antibody treatments is reduced in presence of HRG1.beta.
(McDonagh et al. 2012).
The "epitope of pertuzumab" is the region in the extracellular
domain of HER2 to which the antibody pertuzumab binds. In order to
screen for antibodies which bind to the same epitope as pertuzumab,
a routine cross-blocking assay such as that described in "Ed.
Harlow and David Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, (1988)", can be performed. Alternatively,
epitope mapping can be performed to assess whether the antibody
binds to the pertuzumab epitope of HER2 (e.g. any one or more
residues in the region from about residue 22 to about residue 584
of HER2, inclusive). The binding epitope of pertuzumab comprises
residues from domain II in the extracellular domain of HER2.
Pertuzumab bind to the extracellular domain of HER2 at the junction
of domains I, II and III. See also Franklin, et al., Cancer Cell 5
(2004) 317-328.
The term "HER4," as used herein, refers to any native HER4 from any
vertebrate source, including mammals such as primates (e.g. humans)
and rodents (e.g., mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed HER4 as well as any
form of HER4 that results from processing in the cell. The term
also encompasses naturally occurring variants of HER4, e.g., splice
variants or allelic variants. The amino acid sequence of an
exemplary human HER4 is shown in SEQ ID NO:5. "Human HER4" (also
known as ErbB-4 ERBB4, v-erb-a erythroblastic leukemia viral
oncogene homolog 4, p180erbB4 avian erythroblastic leukemia viral
(v-erb-b2) oncogene homolog 4; SEQ ID NO:5) is a single-pass type I
transmembrane protein with multiple furin-like cysteine rich
domains, a tyrosine kinase domain, a phosphotidylinositol-3 kinase
binding site and a PDZ domain binding motif (Plowman G D, wt al,
PNAS 90:1746-50(1993); Zimonjic D B, et al, Oncogene
10:1235-7(1995); Culouscou J M, et al, J. Biol. Chem.
268:18407-10(1993)). The protein binds to and is activated by
neuregulins-2 and -3, heparin-binding EGF-like growth factor and
betacellulin. Ligand binding induces a variety of cellular
responses including mitogenesis and differentiation. Multiple
proteolytic events allow for the release of a cytoplasmic fragment
and an extracellular fragment. Mutations in this gene have been
associated with cancer. Alternatively spliced variants which encode
different protein isoforms have been described; however, not all
variants have been fully characterized.
As used herein, "treatment" (and grammatical variations thereof
such as "treat" or "treating") refers to clinical intervention in
an attempt to alter the natural course of the individual being
treated, and can be performed either for prophylaxis or during the
course of clinical pathology. Desirable effects of treatment
include, but are not limited to, preventing occurrence or
recurrence of disease, alleviation of symptoms, diminishment of any
direct or indirect pathological consequences of the disease,
preventing metastasis, decreasing the rate of disease progression,
amelioration or palliation of the disease state, and remission or
improved prognosis. In some embodiments, antibodies of the
invention are used to delay development of a disease or to slow the
progression of a disease.
The term "variable region" or "variable domain" refers to the
domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. Kuby
Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano, S. et al., J. Immunol. 150
(1993) 880-887; Clackson, T. et al., Nature 352 (1991)
624-628).
The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
II. Compositions and Methods
In one aspect, the invention is based, in part, on the finding that
using the beta-hairpins of HER3 (and optionally HER4) functionally
presented in a 3-dimensional orientation within SlyD scaffolds (see
e.g FIG. 2, and the polypeptides of SEQ ID NO. 13, and 17 to 24) it
was possible to select antibodies which are specific for the
beta-hairpin of HER3 (and HER4). They are used together with
antibodies against HER2, specifically to the domain II of human
HER2, to generate bispecific antibody that to human HER3 and that
binds to human HER2, wherein the antibody binds within an amino
acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1; beta-hairpin of
human HER3) to human HER3 and which binds to domain II of human
HER2 (SEQ ID NO: 59).
The bispecific antibodies of the invention are useful, e.g., for
the diagnosis or treatment of cancer.
A. Exemplary Bispecific HER3/HER2 Antibodies
The invention provides an isolated bispecific antibody that binds
to human HER3 and that binds to human HER2, wherein the antibody
binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) which is comprised in a polypeptide selected from the group
consisting of:
TABLE-US-00006 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3.
The invention provides an isolated bispecific antibody that binds
to human HER3 and that binds to human HER2,
wherein the antibody binds within an amino acid sequence of
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a
polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3).
The invention provides an isolated bispecific antibody that binds
to the beta-hairpin of human HER3 (SEQ ID NO: 1) and that binds to
domain II of human HER2 (SEQ ID NO: 59).
The invention provides an isolated bispecific antibody that binds
to human HER3 and that binds to human HER2, wherein the antibody
binds to human HER3 within an amino acid sequence of
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a
polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) and wherein the
antibody binds to domain II of human HER2 (SEQ ID NO: 59).
The invention provides an isolated bispecific antibody that binds
to the polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) which
comprises the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and which antibody binds to domain II of human HER2 (SEQ ID NO:
59).
The invention provides an isolated bispecific antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to the same epitope on human HER2 as pertuzumab.
The invention provides an isolated bispecific antibody that binds
to the polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) which
comprises the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and which antibody binds to the same epitope on human HER2 as
pertuzumab.
The invention provides an isolated bispecific antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that competes for binding to human HER2 with pertuzumab.
The invention provides an bispecific isolated antibody that binds
to the polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) which
comprises the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and which antibody competes for binding to human HER2 with
pertuzumab.
The invention provides an bispecific isolated antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to human HER2 and comprises all six heavy and light
chains HVRs of pertuzumab (SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:
62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65).
The invention provides an bispecific isolated antibody that binds
to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1)
and that binds to human HER2 and comprises the VH and VL of
pertuzumab (SEQ ID NO: 66 and SEQ ID NO. 67)).
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein binds also to human HER4 (and is then
designated as multispecific HER3/HER2 antibody antibody).
In one embodiment of the invention the multispecific HER3/HER2
antibody as described herein binds also to the beta-hairpin of
human HER4 PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).
In one embodiment of the invention the multispecific HER3/HER2
antibody as described herein binds also to the polypeptide of SEQ
ID NO: 22 (TtSlyDcys-Her4) which comprises the amino acid sequence
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).
An example antibody which binds to the beta-hairpin of human HER3
and also binds to human HER4, to the beta-hairpin of human HER4
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) and to the polypeptide of SEQ ID
NO: 22 (TtSlyDcys-Her4) which comprises the amino acid sequence
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) is antibody comprising the VH of
SEQ ID NO: 31 (heavy chain variable domain VH, M-05-74) and the VL
of SEQ ID NO: 32 (light chain variable domain VL, M-05-74).
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein does not crossreact with human
HER4.
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein does not crossreact with the
beta-hairpin of human HER4 PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein does not crossreact with the
polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) which comprises the
amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2).
An example antibody which binds to the beta-hairpin of human HER3
and does not crossreact with (does not bind to) human HER4, to the
beta-hairpin of human HER4 PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) and to
the polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4) which comprises
the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2) is
antibody comprising the VH of SEQ ID NO: 51 (heavy chain variable
domain VH, <Her3> M-08-11) and the VL of SEQ ID NO: 52 (light
chain variable domain VL, <Her3> M-08-11).
The invention provides an bispecific isolated antibody a) that
binds to human HER3 and comprises the heavy chain HVRs of SEQ ID
NO: 25 heavy chain HVR-H1, M-05-74, SEQ ID NO: 26 heavy chain
HVR-H2, M-05-74, and SEQ ID NO: 27 heavy chain HVR-H3, M-05-74, and
comprises the light chain heavy chain HVRs of SEQ ID NO: 28 light
chain HVR-L1, M-05-74, SEQ ID NO: 29 light chain HVR-L2, M-05-74,
and SEQ ID NO: 30 light chain HVR-L3, M-05-74; and b) that binds to
human HER2 and comprises the heavy chain HVRs of SEQ ID NO: 60
heavy chain HVR-H1, pertuzumab, SEQ ID NO: 61 heavy chain HVR-H2
pertuzumab, SEQ ID NO: 62 heavy chain HVR-H3, pertuzumab, and
comprises the light chain heavy chain HVRs of SEQ ID NO: 63 light
chain HVR-L1, pertuzumab, SEQ ID NO: 64 light chain HVR-L2,
pertuzumab, and SEQ ID NO: 65 light chain HVR-L3 pertuzumab.
The invention provides an bispecific isolated antibody a) that
binds to human HER3 and comprises i) a variable heavy chain domain
VH with the amino acid sequence of SEQ ID NO:33 and a variable
light chain domain VL with the amino acid sequence of SEQ ID NO:41,
ii) a variable heavy chain domain VH with the amino acid sequence
of SEQ ID NO:33 and a variable light chain domain VL with the amino
acid sequence of SEQ ID NO:39, or iii) a variable heavy chain
domain VH with the amino acid sequence of SEQ ID NO:33 and a
variable light chain domain VL with the amino acid sequence of SEQ
ID NO:42; and b) that binds to human HER2 and a variable heavy
chain domain VH with the amino acid sequence of SEQ ID NO:66 and a
variable light chain domain VL with the amino acid sequence of SEQ
ID NO:67.
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein wherein the bispecific antibody is
bivalent.
In one embodiment of the invention the bispecific HER3/HER2
antibody as described herein has one or more of the following
properties (either alone or in any combination): the antibody a)
binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) which is comprised in a polypeptide selected from the group
consisting of:
TABLE-US-00007 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3;
b) binds to a polypeptide selected from the group consisting
of:
TABLE-US-00008 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3;
c) inhibits the heterodimerisation of HER3/HER2 heterodimers in
MCF-7 cells in a HER3/HER2 coprecipitation assay; d) shows tumor
growth inhibitory activity in vivo; e) binds with an affinity of a
KD value .ltoreq.1.times.10-8 M to HER3-ECD (in one embodiment with
a KD value of 1.times.10-8 M to 1.times.10-13 M; (in one embodiment
with a KD value of 1.times.10-9 M to 1.times.10-13 M); f) binds
with an affinity of a KD value .ltoreq.1.times.10-8 M to HER2-ECD
(in one embodiment with a KD value of 1.times.10-8 M to
1.times.10-13 M; (in one embodiment with a KD value of 1.times.10-9
M to 1.times.10-13 M.
In one preferred embodiment the antibody is of IgG1 or IgG4
isotype. In one preferred embodiment the antibody comprises
constant domains of human origin (human constant domains.). Typical
human constant regions within the meaning of the present invention
comprising the respective human constant domains have the amino
acid sequences of SEQ ID NO: 53 to SEQ ID NO:58 (which are partly
comprising amino acid substitutions).
1. Antibody Affinity
In certain embodiments, an antibody provided herein has a
dissociation constant KD of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9M to 10.sup.-13 M).
In one preferred embodiment, KD is measured using surface plasmon
resonance assays using a BIACORE.RTM.) at 25.degree. C. with
immobilized antigen CM5 chips at .about.10 response units (RU).
Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE,
Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on or ka) and dissociation
rates (k.sub.off or kd) are calculated using a simple one-to-one
Langmuir binding model (BIACORE.RTM. Evaluation Software version
3.2) by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant KD is calculated
as the ratio kd/ka (k.sub.off/k.sub.on.) See, e.g., Chen, Y. et
al., J. Mol. Biol. 293 (1999) 865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
2. Antibody Fragments
In certain embodiments, an antibody provided herein is an antibody
fragment. Antibody fragments include, but are not limited to, Fab,
Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and other
fragments described below. For a review of certain antibody
fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134.
For a review of scFv fragments, see, e.g., Plueckthun, A., In; The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046.
Diabodies are antibody fragments with two antigen-binding sites
that may be bivalent or bispecific. See, for example, EP 0 404 097;
WO 1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134;
and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448. Triabodies and tetrabodies are also described in Hudson,
P. J. et al., Nat. Med. 9 (20039 129-134).
Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy chain variable domain or all or a portion of
the light chain variable domain of an antibody. In certain
embodiments, a single-domain antibody is a human single-domain
antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No.
6,248,516 B1).
Antibody fragments can be made by various techniques, including but
not limited to proteolytic digestion of an intact antibody as well
as production by recombinant host cells (e.g. E. coli or phage), as
described herein.
In one preferred embodiment the antibody fragment is a Fab
fragment. In one preferred embodiment the antibody fragment (in
case constant domains are contained in the fragmant) comprises
constant domains of human origin (human constant domains.).
3. Chimeric and Humanized Antibodies
In certain embodiments, an antibody provided herein is a chimeric
antibody. Certain chimeric antibodies are described, e.g., in U.S.
Pat. No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad.
Sci. USA 81 (1984) 6851-6855). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
Humanized antibodies and methods of making them are reviewed, e.g.,
in Almagro, J. C. and Fransson, J., Front. Biosci. 13 (2008)
1619-1633, and are further described, e.g., in Riechmann, I. et
al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad.
Sci. USA 86 (1989) 10029-10033; U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri, S. V. et al.,
Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan,
E. A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing");
Dall'Acqua, W. F. et al., Methods 36 (2005) 43-60 (describing "FR
shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and
Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the
"guided selection" approach to FR shuffling). Morea, V., et al.,
Methods, Vol 20, Issue 3 (2000) 267-279) and WO2004/006955
(approach via canonical structures).
4. Human Antibodies
In certain embodiments, an antibody provided herein is a human
antibody. Human antibodies can be produced using various techniques
known in the art. Human antibodies are described generally in van
Dijk, M. A. and van de Winkel, J. G., Curr. Opin. Pharmacol. 5
(2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008)
450-459.
Human antibodies may be prepared by administering an immunogen to a
transgenic animal that has been modified to produce intact human
antibodies or intact antibodies with human variable regions in
response to antigenic challenge. Such animals typically contain all
or a portion of the human immunoglobulin loci, which replace the
endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
N., Nat. Biotech. 23 (2005) 1117-1125. See also, e.g., U.S. Pat.
Nos. 6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology;
U.S. Pat. No. 5,770,429 describing HuMab.RTM. technology; U.S. Pat.
No. 7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VelociMouse.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
Human antibodies can also be made by hybridoma-based methods. Human
myeloma and mouse-human heteromyeloma cell lines for the production
of human monoclonal antibodies have been described. (See, e.g.,
Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et
al., Monoclonal Antibody Production Techniques and Applications,
Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et
al., J. Immunol. 147 (1991) 86-95) Human antibodies generated via
human B-cell hybridoma technology are also described in Li, J. et
al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Additional
methods include those described, for example, in U.S. Pat. No.
7,189,826 (describing production of monoclonal human IgM antibodies
from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006)
265-268 (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers, H. P.
and Brandlein, S., Histology and Histopathology 20 (2005) 927-937
and Vollmers, H. P. and Brandlein, S., Methods and Findings in
Experimental and Clinical Pharmacology 27 (2005) 185-191.
Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
5. Library-Derived Antibodies
Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in
Molecular Biology 178 (2001) 1-37 and further described, e.g., in
the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T.
et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol.
Biol. 222 (1992) 581-597; Marks, J. D. and Bradbury, A., Methods in
Molecular Biology 248 (2003) 161-175; Sidhu, S. S. et al., J. Mol.
Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol. Biol. 340
(2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101
(2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284
(2004) 119-132.
In certain phage display methods, repertoires of VH and VL genes
are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter, G. et al., Ann.
Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths, A. D. et al., EMBO J. 12
(1993) 725-734. Finally, naive libraries can also be made
synthetically by cloning non-rearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.
Biol. 227 (1992) 381-388. Patent publications describing human
antibody phage libraries include, for example: U.S. Pat. No.
5,750,373, and US Patent Publication Nos. 2005/0079574,
2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,
2007/0237764, 2007/0292936, and 2009/0002360.
Antibodies or antibody fragments isolated from human antibody
libraries are considered human antibodies or human antibody
fragments herein.
6. Multispecific Antibodies
In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for HER3/HER4 and
the other is for any other antigen.
Multispecific antibodies may also be used to localize cytotoxic
agents to cells which express HER3 and/or HER2 (and HER4).
Bispecific or multispecific antibodies can be prepared as full
length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not
limited to, recombinant co-expression of two immunoglobulin heavy
chain-light chain pairs having different specificities (see
Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO
93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659),
and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.
5,731,168). Multi-specific antibodies may also be made by
engineering electrostatic steering effects for making antibody
Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or
more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,
and Brennan, M. et al., Science 229 (1985) 81-83); using leucine
zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.
A. et al., J. Immunol. 148 (1992) 1547-1553; using "diabody"
technology for making bispecific antibody fragments (see, e.g.,
Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448); and using single-chain Fv (sFv) dimers (see, e.g.
Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing
trispecific antibodies as described, e.g., in Tutt, A. et al., J.
Immunol. 147 (1991) 60-69).
Engineered antibodies with three or more functional antigen binding
sites, including "Octopus antibodies," are also included herein
(see, e.g. US 2006/0025576).
The antibody or fragment herein also includes a "Dual Acting Fab"
or "DAF" comprising an antigen binding site that binds to HER3 as
well as another, different antigen (see, US 2008/0069820, for
example).
The antibody or fragment herein also includes multispecific
antibodies described in WO 2009/080251, WO 2009/080252, WO
2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO
2010/136172, WO 2010/145792, and WO 2010/145793.
7. Antibody Variants
In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
a) Substitution, Insertion, and Deletion Variants
In certain embodiments, antibody variants having one or more amino
acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of "preferred
substitutions". More substantial changes are provided in Table 1
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes.
Amino acid substitutions may be introduced into an antibody of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00009 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain
properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp,
Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain
orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.
One type of substitutional variant involves substituting one or
more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
Alterations (e.g., substitutions) may be made in HVRs, e.g., to
improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or
SDRs (a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom, H. R. et al. in Methods in Molecular Biology 178 (2002)
1-37. In some embodiments of affinity maturation, diversity is
introduced into the variable genes chosen for maturation by any of
a variety of methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may
occur within one or more HVRs so long as such alterations do not
substantially reduce the ability of the antibody to bind antigen.
For example, conservative alterations (e.g., conservative
substitutions as provided herein) that do not substantially reduce
binding affinity may be made in HVRs. Such alterations may be
outside of HVR "hotspots" or SDRs. In certain embodiments of the
variant VH and VL sequences provided above, each HVR either is
unaltered, or contains no more than one, two or three amino acid
substitutions.
A useful method for identification of residues or regions of an
antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, B. C. and Wells,
J. A., Science 244 (1989) 1081-1085. In this method, a residue or
group of target residues (e.g., charged residues such as arg, asp,
his, lys, and glu) are identified and replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to
determine whether the interaction of the antibody with antigen is
affected. Further substitutions may be introduced at the amino acid
locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure
of an antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
b) Glycosylation Variants
In certain embodiments, an antibody provided herein is altered to
increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright, A.
and Morrison, S. L., TIBTECH 15 (1997) 26-32. The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GlcNAc), galactose, and sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US 2003/0157108; US 2004/0093621. Examples of publications related
to "defucosylated" or "fucose-deficient" antibody variants include:
US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140;
Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249;
Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.
Examples of cell lines capable of producing defucosylated
antibodies include Lec13 CHO cells deficient in protein
fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986)
533-545; US 2003/0157108; and WO 2004/056312, especially at Example
11), and knockout cell lines, such as alpha-1,6-fucosyltransferase
gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et
al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al.,
Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).
Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US
2005/0123546. Antibody variants with at least one galactose residue
in the oligosaccharide attached to the Fc region are also provided.
Such antibody variants may have improved CDC function. Such
antibody variants are described, e.g., in WO 1997/30087; WO
1998/58964; and WO 1999/22764.
c) Fc Region Variants
In certain embodiments, one or more amino acid modifications may be
introduced into the Fc region of an antibody provided herein,
thereby generating an Fc region variant. The Fc region variant may
comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions.
In certain embodiments, the invention contemplates an antibody
variant that possesses some but not all effector functions, which
make it a desirable candidate for applications in which the half
life of the antibody in vivo is important yet certain effector
functions (such as complement and ADCC) are unnecessary or
deleterious. In vitro and/or in vivo cytotoxicity assays can be
conducted to confirm the reduction/depletion of CDC and/or ADCC
activities. For example, Fc receptor (FcR) binding assays can be
conducted to ensure that the antibody lacks Fc.gamma.R binding
(hence likely lacking ADCC activity), but retains FcRn binding
ability. The primary cells for mediating ADCC, NK cells, express
Fc(RIII only, whereas monocytes express FcgammaRI, FcgammaRII and
FcgammaRIII. FcR expression on hematopoietic cells is summarized in
Table 3 on page 464 of Ravetch, J. V. and Kinet, J. P., Annu. Rev.
Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al., Proc.
Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al.,
Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); U.S. Pat. No.
5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987)
1351-1361). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.; and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g., in an
animal model such as that disclosed in Clynes, R. et al., Proc.
Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays may also
be carried out to confirm that the antibody is unable to bind C1q
and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA
in WO 2006/029879 and
WO 2005/100402. To assess complement activation, a CDC assay may be
performed (see, for example, Gazzano-Santoro, H. et al., J.
Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101
(2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103
(2004) 2738-2743). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006:
1759-1769).
Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
Certain antibody variants with improved or diminished binding to
FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO
2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)
6591-6604)
In certain embodiments, an antibody variant comprises an Fc region
with one or more amino acid substitutions which improve ADCC, e.g.,
substitutions at positions 298, 333, and/or 334 of the Fc region
(EU numbering of residues).
In some embodiments, alterations are made in the Fc region that
result in altered (i.e., either improved or diminished) C1q binding
and/or Complement Dependent Cytotoxicity (CDC), e.g., as described
in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie, E. E. et
al., J. Immunol. 164 (2000) 4178-4184.
Antibodies with increased half lives and improved binding to the
neonatal Fc receptor (FcRn), which is responsible for the transfer
of maternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117
(1976) 587-593, and Kim, J. K. et al., J. Immunol. 24 (1994)
2429-2434), are described in US 2005/0014934. Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740;
U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning
other examples of Fc region variants.
d) Cysteine Engineered Antibody Variants
In certain embodiments, it may be desirable to create cysteine
engineered antibodies, e.g., "thioMAbs," in which one or more
residues of an antibody are substituted with cysteine residues. In
particular embodiments, the substituted residues occur at
accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
e) Antibody Derivatives
In certain embodiments, an antibody provided herein may be further
modified to contain additional non-proteinaceous moieties that are
known in the art and readily available. The moieties suitable for
derivatization of the antibody include but are not limited to water
soluble polymers. Non-limiting examples of water soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
In another embodiment, conjugates of an antibody and
non-proteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the non-proteinaceous
moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad.
Sci. USA 102 (2005) 11600-11605). The radiation may be of any
wavelength, and includes, but is not limited to, wavelengths that
do not harm ordinary cells, but which heat the non-proteinaceous
moiety to a temperature at which cells proximal to the
antibody-non-proteinaceous moiety are killed.
B. Recombinant Methods and Compositions
Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an bispecific HER3/HER2
antibody described herein is provided. Such nucleic acid may encode
an amino acid sequence comprising the VL and/or an amino acid
sequence comprising the VH of the antibody (e.g., the light and/or
heavy chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an bispecific
HER3/HER2 antibody is provided, wherein the method comprises
culturing a host cell comprising a nucleic acid encoding the
antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
For recombinant production of an bispecific HER3/HER2 antibody,
nucleic acid encoding an antibody, e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
Suitable host cells for cloning or expression of antibody-encoding
vectors include prokaryotic or eukaryotic cells described herein.
For example, antibodies may be produced in bacteria, in particular
when glycosylation and Fc effector function are not needed. For
expression of antibody fragments and polypeptides in bacteria, see,
e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also
Charlton, K. A., In: Methods in Molecular Biology, Vol. 248, Lo, B.
K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,
describing expression of antibody fragments in E. coli.) After
expression, the antibody may be isolated from the bacterial cell
paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are suitable cloning or expression hosts for
antibody-encoding vectors, including fungi and yeast strains whose
glycosylation pathways have been "humanized," resulting in the
production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22
(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006)
210-215.
Suitable host cells for the expression of glycosylated antibody are
also derived from multicellular organisms (invertebrates and
vertebrates). Examples of invertebrate cells include plant and
insect cells. Numerous baculoviral strains have been identified
which may be used in conjunction with insect cells, particularly
for transfection of Spodoptera frugiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., U.S.
Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian
cell lines that are adapted to grow in suspension may be useful.
Other examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney
line (293 or 293 cells as described, e.g., in Graham, F. L. et al.,
J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK);
mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.
P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1);
African green monkey kidney cells (VERO-76); human cervical
carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat
liver cells (BRL 3A); human lung cells (W138); human liver cells
(Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as
described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci.
383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR.sup.- CHO cells (Urlaub, G. et al., Proc.
Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines
such as Y0, NS0 and Sp2/0. For a review of certain mammalian host
cell lines suitable for antibody production, see, e.g., Yazaki, P.
and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
C. Assays
Bispecific HER3/HER2 (and their parent anti-HER3 and anti-HER2)
antibodies provided herein may be identified, screened for, or
characterized for their physical/chemical properties and/or
biological activities by various assays known in the art.
Disclosed is a method for selecting for an antibody that binds to
human HER3 for use in generating bispecific HER3/HER2 antibodies
wherein the anti-HER3 antibody binds within an amino acid sequence
of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) of human HER3; wherein
a) at least one polypeptide selected from the group consisting
of:
TABLE-US-00010 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3,
which comprises the amino acid sequence of SEQ ID NO:1; are used
(in a binding assay) to select antibodies, which show binding to
the at least one polypeptide under a) and thereby selecting an
antibody that binds within an amino acid sequence of
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) (within human HER3).
In one embodiment the selection method further comprises a step
wherein the selected antibodies are counterscreened with the
polypeptides (tested for binding to the polypeptides) selected from
the group consisting of:
TABLE-US-00011 SEQ ID NO: 14 TtSlyD-Wildtype SEQ ID NO: 15
TtSlyDcas SEQ ID NO: 16 TgSlyD.DELTA.IF
to confirm that the selected antibodies do not bind to the
polypeptide scaffolds which are not comprising amino acid sequence
of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1). 1. Binding Assays and Other
Assays
In one aspect, an antibody of the invention is tested for its
antigen binding activity, e.g., by known methods such as ELISA,
Western blot, including surface plasmon resonance (e.g. BIACORE),
etc.
In another aspect, competition assays may be used to identify an
antibody that competes with M-05-74 for binding to HER3 (and/or to
HER4) and also to identify an antibody that competes with
pertuzumab for binding to HER2. In certain embodiments, such a
competing antibody binds to the same epitope (e.g., a linear or a
conformational epitope) that is bound by M-05-74. Detailed
exemplary methods for mapping an epitope to which an antibody binds
are provided in Morris, G. E. (ed.), Epitope Mapping Protocols, In:
Methods in Molecular Biology, Vol. 66, Humana Press, Totowa, N.J.
(1996). Further methods are described in detail in Example 4 using
the CelluSpot.TM. technology.
In an exemplary competition assay, immobilized HER3(/HER4), or to
HER2 is incubated in a solution comprising a first labeled antibody
that binds to HER3(/HER4), or to HER2, respectively (e.g., M-05-74
or pertuzumab) and a second unlabeled antibody that is being tested
for its ability to compete with the first antibody for binding to
HER3(/HER4), or to HER2. The second antibody may be present in a
hybridoma supernatant. As a control, immobilized HER3 or HER4 is
incubated in a solution comprising the first labeled antibody but
not the second unlabeled antibody. After incubation under
conditions permissive for binding of the first antibody to
HER3(/HER4), or to HER2, excess unbound antibody is removed, and
the amount of label associated with immobilized HER3(/HER4), or to
HER2 is measured. If the amount of label associated with
immobilized HER3(/HER4), or to HER2 is substantially reduced in the
test sample relative to the control sample, then that indicates
that the second antibody is competing with the first antibody for
binding to HER3(/HER4), or to HER2. See Harlow, E. and Lane, D.,
Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1988).
2. Activity Assays
In one aspect, assays are provided for identifying bispecific
HER3/HER2 antibodies thereof having biological activity. Biological
activity may include, e.g., inhibition of HER3 and/or HER2
phosphorylation, inhibition of cancer cell proliferation of HER3
and/or HER2 (and/or HER4) expressing or overexpressing cancer
cells, inhibition of HER3/HER2 heterodimerization, (time-dependent)
internalization via FACS assay, in vivo tumor growth inhibition in
xenograft animal (e.g. mouse or rat) models with xenografted HER3
and/or HER2 (and/or HER4) expressing or overexpressing cancer
cells. Antibodies having such biological activity in vivo and/or in
vitro are also provided.
In certain embodiments, an antibody of the invention is tested for
such biological activity. Exemplary vitro or in vivo assays for
specified biological activities are described in Example 2e, 3, 5
to 9, and 11 or 17.
D. Immunoconjugates
The invention also provides immunoconjugates comprising an
anti-HER3/HER4 antibody described herein conjugated to one or more
cytotoxic agents, such as chemotherapeutic agents or drugs, growth
inhibitory agents, toxins (e.g., protein toxins, enzymatically
active toxins of bacterial, fungal, plant, or animal origin, or
fragments thereof), or radioactive isotopes.
In one embodiment, an immunoconjugate is an antibody-drug conjugate
(ADC) in which an antibody is conjugated to one or more drugs,
including but not limited to a maytansinoid (see U.S. Pat. Nos.
5,208,020, 5,416,064 and EP 0 425 235 B1); an auristatin such as
monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see
U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); a dolastatin;
a calicheamicin or derivative thereof (see U.S. Pat. Nos.
5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,
5,773,001, and 5,877,296; Hinman, L. M. et al., Cancer Res. 53
(1993) 3336-3342; and Lode, H. N. et al., Cancer Res. 58 (1998)
2925-2928); an anthracycline such as daunomycin or doxorubicin (see
Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523; Jeffrey, S.
C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362; Torgov, M.
Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. et al.,
Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, G. M. et
al., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H.
D. et al., J. Med. Chem. 45 (20029 4336-4343; and U.S. Pat. No.
6,630,579); methotrexate; vindesine; a taxane such as docetaxel,
paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene;
and CC1065.
In another embodiment, an immunoconjugate comprises an antibody as
described herein conjugated to an enzymatically active toxin or
fragment thereof, including but not limited to diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
In another embodiment, an immunoconjugate comprises an antibody as
described herein conjugated to a Pseudomonas exotoxin A or variants
thereof. Pseudomonas exotoxin A or variants thereof are described
e.g in WO2011/32022, WO2009/32954, WO2007/031741, WO2007/016150,
WO2005/052006 and Liu W, et al, PNAS 109 (2012) 11782-11787.
In another embodiment, an immunoconjugate comprises an antibody as
described herein conjugated to a radioactive atom to form a
radioconjugate. A variety of radioactive isotopes are available for
the production of radioconjugates. Examples include At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu.
When the radioconjugate is used for detection, it may comprise a
radioactive atom for scintigraphic studies, for example TC.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, MRI), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
Conjugates of an antibody and cytotoxic agent may be made a) either
using recombination expression techniques (e.g for the expression
of amino acid sequence based toxines fused to a Fab or Fv antibody
fragment e.g. in E. coli) or b) using polypeptide coupling
techniques (like sortase enzyme based coupling of amino acid
sequence based toxines to a Fab or Fv antibody fragment) or c)
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta, E. S. et al.,
Science 238 (1987) 1098-1104. Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triamine pentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO 94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari, R. V. et al., Cancer Res. 52 (1992) 127-131; U.S.
Pat. No. 5,208,020) may be used.
The immunuoconjugates or ADCs herein expressly contemplate, but are
not limited to such conjugates prepared with cross-linker reagents
including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC,
MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
E. Methods and Compositions for Diagnostics and Detection
In certain embodiments, any of the bispecific HER3/HER2 antibodies
provided herein is useful for detecting the presence of HER3 and/or
HER4, respectively in a biological sample. The term "detecting" as
used herein encompasses quantitative or qualitative detection. In
certain embodiments, a biological sample comprises a cell or
tissue, such as tumor tissues.
In one embodiment, an bispecific HER3/HER2 antibody for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of HER3 or HER2, respectively,
in a biological sample is provided. In certain embodiments, the
method comprises contacting the biological sample with an
bispecific HER3/HER2 antibody as described herein under conditions
permissive for binding of the anti-bispecific HER3/HER2 antibody to
HER3 or HER2, respectively, and detecting whether a complex is
formed between the anti-bispecific HER3/HER2 antibody and HER3 or
HER2, respectively. Such method may be an in vitro or in vivo
method. In one embodiment, an bispecific HER3/HER2 antibody is used
to select subjects eligible for therapy with an the bispecific
HER3/HER2 antibodies antibody, e.g. where HER3 and HER2,
respectively are both biomarkers for selection of patients.
Exemplary disorders that may be diagnosed using an antibody of the
invention include cancer.
In certain embodiments, labeled bispecific HER3/HER2 antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
F. Pharmaceutical Formulations
Pharmaceutical formulations of an bispecific HER3/HER2 antibody as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences, 16th
edition, Osol, A. (ed.) (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as
poly(vinylpyrrolidone); amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
interstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rhuPH20, are described in US Patent Publication
Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is
combined with one or more additional glycosaminoglycanases such as
chondroitinases.
Exemplary lyophilized antibody formulations are described in U.S.
Pat. No. 6,267,958. Aqueous antibody formulations include those
described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter
formulations including a histidine-acetate buffer.
The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated.
Active ingredients may be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methyl methacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed.) (1980).
Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semi-permeable matrices
of solid hydrophobic polymers containing the antibody, which
matrices are in the form of shaped articles, e.g. films, or
microcapsules.
The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
G. Therapeutic Methods and Compositions
Any of the bispecific HER3/HER2 antibodies or immunoconjugates of
the anti-bispecific HER3/HER2 antibodies conjugated to a cytotoxic
agent, provided herein may be used in therapeutic methods.
In one aspect, an bispecific HER3/HER2 antibody or immunoconjugate
of the anti-v antibody conjugated to a cytotoxic agent for use as a
medicament is provided. In further aspects, an bispecific HER3/HER2
antibody or immunoconjugate of the bispecific HER3/HER2 antibody
conjugated to a cytotoxic agent for use in treating cancer is
provided. In certain embodiments, an bispecific HER3/HER2 antibody
or immunoconjugates of the bispecific HER3/HER2 antibody conjugated
to a cytotoxic agent for use in a method of treatment is provided.
In certain embodiments, the invention provides an bispecific
HER3/HER2 antibody or immunoconjugate of the bispecific HER3/HER2
antibody conjugated to a cytotoxic agent for use in a method of
treating an individual having cancer comprising administering to
the individual an effective amount of the bispecific HER3/HER2
antibody or the immunoconjugate of the bispecific HER3/HER2
antibody conjugated to a cytotoxic agent. In further embodiments,
the invention provides an bispecific HER3/HER2 antibody or
immunoconjugate of the bispecific HER3/HER2 antibody conjugated to
a cytotoxic agent for use in inducing apoptosis in a cancer cell/or
inhibiting cancer cell proliferation. In certain embodiments, the
invention provides an bispecific HER3/HER2 antibody or
immunoconjugate of the bispecific HER3/HER2 antibody conjugated to
a cytotoxic agent for use in a method of inducing apoptosis in a
cancer cell/or inhibiting cancer cell proliferation in an
individual comprising administering to the individual an effective
of the bispecific HER3/HER2 antibody or immunoconjugate of the
bispecific HER3/HER2 antibodies conjugated to a cytotoxic agent to
induce apoptosis in a cancer cell/or to inhibit cancer cell
proliferation. An "individual" according to any of the above
embodiments is preferably a human.
In a further aspect, the invention provides for the use of an
bispecific HER3/HER2 antibody or an immunoconjugate of the
bispecific HER3/HER2 antibody conjugated to a cytotoxic agent in
the manufacture or preparation of a medicament. In one embodiment,
the medicament is for treatment of cancer. In a further embodiment,
the medicament is for use in a method of treating cancer comprising
administering to an individual having cancer an effective amount of
the medicament. In a further embodiment, the medicament is for
inducing apoptosis in a cancer cell/or inhibiting cancer cell
proliferation. In a further embodiment, the medicament is for use
in a method of inducing apoptosis in a cancer cell/or inhibiting
cancer cell proliferation in an individual suffering from cancer
comprising administering to the individual an amount effective of
the medicament to induce apoptosis in a cancer cell/or to inhibit
cancer cell proliferation. An "individual" according to any of the
above embodiments may be a human.
In a further aspect, the invention provides a method for treating
cancer. In one embodiment, the method comprises administering to an
individual having cancer an effective amount of an bispecific
HER3/HER2 antibody. An "individual" according to any of the above
embodiments may be a human.
In a further aspect, the invention provides a method for inducing
apoptosis in a cancer cell/or inhibiting cancer cell proliferation
in an individual suffering from cancer. In one embodiment, the
method comprises administering to the individual an effective
amount of an bispecific HER3/HER2 antibody or an immunoconjugate of
the bispecific HER3/HER2 antibody conjugated to a cytotoxic
compound to induce apoptosis in a cancer cell/or to inhibit cancer
cell proliferation in the individual suffering from cancer. In one
embodiment, an "individual" is a human.
In a further aspect, the invention provides pharmaceutical
formulations comprising any of the bispecific HER3/HER2 antibodies
provided herein, e.g., for use in any of the above therapeutic
methods. In one embodiment, a pharmaceutical formulation comprises
any of the bispecific HER3/HER2 antibodies provided herein and a
pharmaceutically acceptable carrier.
An antibody of the invention (and any additional therapeutic agent)
can be administered by any suitable means, including parenteral,
intrapulmonary, and intranasal, and, if desired for local
treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
For the prevention or treatment of disease, the appropriate dosage
of an antibody of the invention (when used alone or in combination
with one or more other additional therapeutic agents) will depend
on the type of disease to be treated, the type of antibody, the
severity and course of the disease, whether the antibody is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading
dose of about 4 mg/kg, followed by a weekly maintenance dose of
about 2 mg/kg of the antibody. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
It is understood that any of the above formulations or therapeutic
methods may be carried out using an immunoconjugate of the
invention in place of or in addition to an anti-HER3(and anti-HER4)
antibody.
III. Articles of Manufacture
In another aspect of the invention, an article of manufacture
containing materials useful for the treatment, prevention and/or
diagnosis of the disorders described above is provided. The article
of manufacture comprises a container and a label or package insert
on or associated with the container. Suitable containers include,
for example, bottles, vials, syringes, IV solution bags, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds a composition which is by itself or
combined with another composition effective for treating,
preventing and/or diagnosing the condition and may have a sterile
access port (for example the container may be an intravenous
solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). At least one active agent in the composition is
an antibody of the invention. The label or package insert indicates
that the composition is used for treating the condition of choice.
Moreover, the article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises an antibody of the invention; and (b) a
second container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic
agent. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
It is understood that any of the above articles of manufacture may
include an immunoconjugate of the invention in place of or in
addition to an anti-HER3 (and anti-HER4) antibody.
Description of the Amino Acid Sequences
SEQ ID NO: 1 -Hairpin of human HER3 SEQ ID NO: 2 -Hairpin of human
HER4 SEQ ID NO: 3 human HER3 SEQ ID NO: 4 human HER3 Extracellular
Domain (ECD) SEQ ID NO: 5 human HER4 SEQ ID NO: 6 human HER4
Extracellular Domain (ECD) SEQ ID NO: 7 human HER1 SEQ ID NO: 8
human HER1 Extracellular Domain (ECD) SEQ ID NO: 9 human HER2 SEQ
ID NO: 10 human HER2 Extracellular Domain (ECD) SEQ ID NO: 11 Human
Heregulin fragment (HRG) SEQ ID NO: 12 Human Heregulin .beta.-1
fragment (as provided from Preprotech) SEQ ID NO: 13
TtSlyD-FKBP-Her3 SEQ ID NO: 14 TtSlyD-Wildtype SEQ ID NO: 15
TtSlyDcas SEQ ID NO: 16 TgSlyD.DELTA.IF SEQ ID NO: 17
TtSlyDcas-Her3 SEQ ID NO: 18 TtSlyDcys-Her3 SEQ ID NO: 19
TgSlyDser-Her3 SEQ ID NO: 20 TgSlyDcys-Her3 SEQ ID NO: 21
TtSlyDcas-Her4 SEQ ID NO: 22 TtSlyDcys-Her4 SEQ ID NO: 23
TgSlyDser-Her4 SEQ ID NO: 24 TgSlyDcys-Her4 SEQ ID NO: 25 heavy
chain HVR-H1, M-05-74 SEQ ID NO: 26 heavy chain HVR-H2, M-05-74 SEQ
ID NO: 27 heavy chain HVR-H3, M-05-74 SEQ ID NO: 28 light chain
HVR-L1, M-05-74 SEQ ID NO: 29 light chain HVR-L2, M-05-74 SEQ ID
NO: 30 light chain HVR-L3, M-05-74 SEQ ID NO: 31 heavy chain
variable domain VH, M-05-74 SEQ ID NO: 32 light chain variable
domain VL, M-05-74 SEQ ID NO: 33 humanized variant A of heavy chain
variable domain VH, M-05-74_VH-A SEQ ID NO: 34 humanized variant B
of heavy chain variable domain VH, M-05-74_VH-B SEQ ID NO: 35
humanized variant 3 of heavy chain variable domain VH, M-05-74_VH-C
SEQ ID NO: 36 humanized variant A of heavy chain variable domain
VH, M-05-74_VH-D SEQ ID NO: 37 humanized variant B of heavy chain
variable domain VH, M-05-74_VH-E SEQ ID NO: 38 humanized variant A
of light chain variable domain VL, M-05-74_VL-A SEQ ID NO: 39
humanized variant B of light chain variable domain VL, M-05-74_VL-B
SEQ ID NO: 40 humanized variant C of light chain variable domain
VL, M-05-74_VL-C SEQ ID NO: 41 humanized variant D of light chain
variable domain VL, M-05-74_VL-D SEQ ID NO: 42 humanized variant E
of light chain variable domain VL, M-05-74_VL-E SEQ ID NO:43
binding epitope within -hairpin of human HER3 SEQ ID NO:44 binding
epitope within -hairpin of human HER4 SEQ ID NO:45 Pseudomonas
exotoxin variant PE24LR8M 3_G (including a GGG linker) SEQ ID NO:46
Light chain of M-05-74 (M-05-74_LC) SEQ ID NO:47 Heavy chain of
M-05-74 HC with sortase tag (M-05-74_HC) SEQ ID NO:48 Heavy chain
of M-05-74 HC conjugated to Pseudomonas exotoxin variant PE24LR8M
(Fab-074-PE heavy chain 1) SEQ ID NO:49 Heavy chain of M-05-74 HC
conjugated to Pseudomonas exotoxin variant PE24LR8M (Fab-074-PE
heavy chain 2) as direct PE24LR8M fusion SEQ ID NO: 50 soluble S.
aureus sortase A SEQ ID NO: 51 heavy chain variable domain VH,
<Her3> M-08-11 SEQ ID NO: 52 light chain variable domain VL,
<Her3> M-08-11 SEQ ID NO: 53 human kappa light chain constant
region SEQ ID NO: 54 human lambda light chain constant region SEQ
ID NO: 55 human heavy chain constant region derived from IgG1 SEQ
ID NO: 56 human heavy chain constant region derived from IgG1
mutated on L234A and L235A SEQ ID NO: 57 human heavy chain constant
region derived from IgG1 mutated on L234A, L235A and P329G SEQ ID
NO: 58 human heavy chain constant region derived from IgG4 SEQ ID
NO: 59 domain II of human HER2 SEQ ID NO: 60 heavy chain HVR-H1,
pertuzumab SEQ ID NO: 61 heavy chain HVR-H2 pertuzumab SEQ ID NO:
62 heavy chain HVR-H3, pertuzumab SEQ ID NO: 63 light chain HVR-L1,
pertuzumab SEQ ID NO: 64 light chain HVR-L2, pertuzumab SEQ ID NO:
65 light chain HVR-L3 pertuzumab SEQ ID NO: 66 heavy chain variable
domain VH, pertuzumab SEQ ID NO: 67 light chain variable domain VL,
pertuzumab SEQ ID NO: 68 heavy chain 1, bispecific HER3/HER2
antibody DIB.times.PERT SEQ ID NO: 69 light chain 1, bispecific
HER3/HER2 antibody DIB.times.PERT SEQ ID NO: 70 heavy chain 2,
bispecific HER3/HER2 antibody DIB.times.PERT SEQ ID NO: 71 light
chain 2, bispecific HER3/HER2 antibody DIB.times.PERT
The following examples and figures are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention. The disclosures of all
patent and scientific literature cited herein are expressly
incorporated by reference in their entirety.
In the Following Several Embodiments of the Invention are
Listed
1. Use of at least one polypeptide selected from the group
consisting of:
TABLE-US-00012 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3,
which comprises the amino acid sequence of SEQ ID NO:1; in a method
for selecting an antibody that binds to human HER3 for use in the
generation of a bispecific HER3/HER2 antibody, wherein the HER3
antibody binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT
(SEQ ID NO:1) of human HER3; and such HER3 antibody is then used to
generate a bispecific HER3/HER2 antibody. 2. An isolated bispecific
antibody which to human HER3 and to human HER2, wherein the
antibody binds within an amino acid sequence of PQPLVYNKLTFQLEPNPHT
(SEQ ID NO:1) which is comprised in a polypeptide selected from the
group consisting of:
TABLE-US-00013 SEQ ID NO: 13 TtSlyD-FKBP-Her3, SEQ ID NO: 17
TtSlyDcas-Her3, SEQ ID NO: 18 TtSlyDcys-Her3, SEQ ID NO: 19
TgSlyDser-Her3, and SEQ ID NO: 20 TgSlyDcys-Her3.
3. An isolated bispecific antibody which to human HER3 and to human
HER2, wherein the antibody binds within an amino acid sequence of
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is comprised in a
polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3). 4. An isolated
bispecific antibody that binds to the beta-hairpin of human HER3
(SEQ ID NO: 1) and that binds to domain II of human HER2 (SEQ ID
NO: 59). 5. An isolated bispecific antibody which to human HER3 and
to human HER2, wherein the antibody binds to human HER3 within an
amino acid sequence of PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) which is
comprised in a polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) and
wherein the antibody binds to domain II of human HER2 (SEQ ID NO:
59). 6. An isolated bispecific antibody which antibody binds to the
polypeptide of SEQ ID NO: 18 (TtSlyDcys-Her3) which comprises the
amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and which
antibody binds to domain II of human HER2 (SEQ ID NO: 59). 7. An
isolated bispecific antibodies that binds to the beta-hairpin of
human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and that binds to the
same epitope on human HER2 as pertuzumab. 8. An isolated bispecific
antibody which antibody binds to the polypeptide of SEQ ID NO: 18
(TtSlyDcys-Her3) which comprises the amino acid sequence
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and which antibody binds to the
same epitope on human HER2 as pertuzumab. 9. An isolated bispecific
antibodies that binds to the beta-hairpin of human HER3
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and that competes for binding to
human HER2 with pertuzumab. 10. An isolated bispecific antibody
which antibody binds to the polypeptide of SEQ ID NO: 18
(TtSlyDcys-Her3) which comprises the amino acid sequence
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and which antibody competes for
binding to human HER2 with pertuzumab. 11. An isolated bispecific
antibodies that binds to the beta-hairpin of human HER3
PQPLVYNKLTFQLEPNPHT (SEQ ID NO:1) and that binds to human HER2 and
comprises all six heavy and light chains HVRs of pertuzumab (SEQ ID
NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,
and SEQ ID NO: 65). 12. An isolated bispecific antibodies that
binds to the beta-hairpin of human HER3 PQPLVYNKLTFQLEPNPHT (SEQ ID
NO:1) and that binds to human HER2 and comprises the VH and VL of
pertuzumab (SEQ ID NO: 66 and SEQ ID NO. 67). 13. The bispecific
HER3/HER2 antibody according to any one of the preceding
embodiments that binds also to human HER4. 14. The bispecific
HER3/HER2 antibody according to any one of the preceding
embodiments that binds also to the beta-hairpin of human HER4
PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2). 15. The bispecific HER3/HER2
antibody according to any one of the preceding embodiments that
binds also to the polypeptide of SEQ ID NO: 22 (TtSlyDcys-Her4)
which comprises the amino acid sequence PQTFVYNPTTFQLEHNFNA (SEQ ID
NO:2). 16. The bispecific HER3/HER2 antibody according to any one
of the preceding embodiments that does not crossreact with human
HER4. 17. The bispecific HER3/HER2 antibody according to any one of
the preceding embodiments that does not crossreact with the
beta-hairpin of human HER4 PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2). 18.
The bispecific HER3/HER2 antibody according to any one of the
preceding embodiments that does not crossreact with the polypeptide
of SEQ ID NO: 22 (TtSlyDcys-Her4) which comprises the amino acid
sequence PQTFVYNPTTFQLEHNFNA (SEQ ID NO:2). 19. An isolated
bispecific antibody a) that binds to human HER3 and comprises the
heavy chain HVRs of SEQ ID NO: 25 heavy chain HVR-H1, M-05-74, SEQ
ID NO: 26 heavy chain HVR-H2, M-05-74, and SEQ ID NO: 27 heavy
chain HVR-H3, M-05-74, and comprises the light chain heavy chain
HVRs of SEQ ID NO: 28 light chain HVR-L1, M-05-74, SEQ ID NO: 29
light chain HVR-L2, M-05-74, and SEQ ID NO: 30 light chain HVR-L3,
M-05-74; and b) that binds to human HER2 and comprises the heavy
chain HVRs of SEQ ID NO: 60 heavy chain HVR-H1, pertuzumab, SEQ ID
NO: 61 heavy chain HVR-H2 pertuzumab, SEQ ID NO: 62 heavy chain
HVR-H3, pertuzumab, and comprises the light chain heavy chain HVRs
of SEQ ID NO: 63 light chain HVR-L1, pertuzumab, SEQ ID NO: 64
light chain HVR-L2, pertuzumab, and SEQ ID NO: 65 light chain
HVR-L3 pertuzumab. 20. An isolated bispecific antibody a) that
binds to human HER3 and comprises i) a variable heavy chain domain
VH with the amino acid sequence of SEQ ID NO:33 and a variable
light chain domain VL with the amino acid sequence of SEQ ID NO:41,
ii) a variable heavy chain domain VH with the amino acid sequence
of SEQ ID NO:33 and a variable light chain domain VL with the amino
acid sequence of SEQ ID NO:39, or iii) a variable heavy chain
domain VH with the amino acid sequence of SEQ ID NO:33 and a
variable light chain domain VL with the amino acid sequence of SEQ
ID NO:42; and b) that binds to human HER2 and a variable heavy
chain domain VH with the amino acid sequence of SEQ ID NO:66 and a
variable light chain domain VL with the amino acid sequence of SEQ
ID NO:67. 21. The bispecific HER3/HER2 antibody according to any
one of the preceding embodiments wherein the bispecific antibody is
bivalent. 22. An isolated nucleic acid encoding the bispecific
HER3/HER2 antibody according to any one of the preceding
embodiments. 23. A host cell comprising the nucleic acid of
embodiment 22. 24. A method of producing the bispecific HER3/HER2
antibody according to any one of the preceding embodiments
comprising culturing such host cell so that the antibody is
produced. 25. The method of embodiment 24 which further comprises
recovering such antibody from the host cell. 26. An immunoconjugate
comprising the bispecific HER3/HER2 antibody according to any one
of the preceding embodiments and a cytotoxic agent. 27. A
pharmaceutical formulation comprising The bispecific HER3/HER2
antibody according to any one of the preceding embodiments and a
pharmaceutically acceptable carrier 28. The bispecific HER3/HER2
antibody according to any one of the preceding embodiments for use
as a medicament. 29. The bispecific HER3/HER2 antibody according to
any one of the preceding embodiments, or the immunoconjugate
comprising the bispecific HER3/HER2 antibody and a cytotoxic agent,
for use in treating cancer. 30. The bispecific HER3/HER2 antibody
according to any one of the preceding embodiments for use in
inhibition of HER3/HER2 dimerization and/or HER2/HER2 dimerization.
31. Use of the bispecific HER3/HER2 antibody according to any one
of the preceding embodiments, or an immunoconjugate comprising the
bispecific HER3/HER2 antibody and a cytotoxic agent, in the
manufacture of a medicament. 32. Use of embodiment 31 wherein the
medicament is for treatment of cancer. 33. Use of embodiment 31
wherein the medicament is for the inhibition of HER3/HER2
dimerization and/or HER2/HER2 dimerization. 34. A method of
treating an individual having cancer comprising administering to
the individual an effective amount of the bispecific HER3/HER2
antibody according to any one of the preceding embodiments, or an
immunoconjugate comprising the bispecific HER3/HER2 antibody and a
cytotoxic agent. 35. A method of inhibiting growth of a tumor cell
in an individual suffering from cancer comprising administering to
the individual an effective amount of the bispecific HER3/HER2
antibody according to any one of the preceding embodiments, thereby
inhibiting growth of a tumor cell in the individual.
EXAMPLES
Materials & General Methods
Recombinant DNA Techniques
Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
Gene Synthesis
Desired gene segments were prepared from oligonucleotides made by
chemical synthesis. The 400-1600 bp long gene segments, which were
flanked by singular restriction endonuclease cleavage sites, were
assembled by annealing and ligating oligonucleotides including PCR
amplification and subsequently cloned via the indicated restriction
sites e.g. EcoRI/BlpI or BsmI/XhoI into the expression vectors
described below. The DNA sequences of the subcloned gene fragments
were confirmed by DNA sequencing. Gene synthesis fragments were
ordered according to given specifications at Geneart (Regensburg,
Germany).
DNA Sequence Determination
DNA sequences were determined by double strand sequencing performed
at Sequiserve GmbH (Vaterstetten, Germany).
DNA and Protein Sequence Analysis and Sequence Data Management
Infomax's Vector NT1 Advance suite version 11.5.0 was used for
sequence creation, mapping, analysis, annotation and
illustration.
Example 1
Preparation of Antigen and Screening Proteins--Generation of
Functional -Hairpin HER3 and -Hairpin HER4 Constructs for Selecting
Antibodies Binding to the -Hairpin of HER3 and the -Hairpin of
HER4
To generate functional -Hairpin HER3 and HER4 constructs, the amino
acid sequences of the -Hairpins of HER3 (SEQ ID NO: 1) and HER4
(SEQ ID NO: 2), were grafted into a SlyD polypeptide framework
comprising a FKBP domain. In such constructs the grafted -Hairpins
are freely accessible in contrast to the hidden structure in the
native unactivated conformation of HER3 or HER4 (in the absence of
ligand as e.g. HRG) (see FIGS. 1c and 1d where the -Hairpin of HER3
is hidden).
All fused SlyD polypeptides can be purified and refolded by using
almost identical protocols. E. coli BL21 (DE3) cells transformed
with the particular expression plasmid were grown at 37.degree. C.
in LB medium containing the respective antibiotic for selective
growth (Kanamycin 30 .mu.g/ml, or Ampicillin (100 .mu.g/ml)) to an
OD600 of 1.5, and cytosolic overexpression was induced by adding 1
mM isopropyl- -D-thiogalactoside (IPTG). Three hours after
induction, cells were harvested by centrifugation (20 min at 5,000
g), frozen and stored at -20.degree. C. For cell lysis, the frozen
pellet was resuspended in chilled 50 mM sodium phosphate buffer (pH
8.0) supplemented with 7 M GdmCl and 5 mM imidazole. Thereafter the
suspension was stirred for 2-10 hours on ice to complete cell
lysis. After centrifugation (25,000 g, 1 h) and filtration
(cellulose nitrate membrane, 8.0 .mu.m, 1.2 .mu.m, 0.2 .mu.m), the
lysate was applied onto a Ni-NTA column equilibrated with the lysis
buffer. In the subsequent washing step the imidazole concentration
was raised to 10 mM (in 50 mM sodium phosphate buffer (pH 8.0)
comprising 7 M GdmCl) and 5 mM TCEP was added in order to keep the
thiol moieties in a reduced form and to prevent premature disulfide
bridging. At least 15 to 20 volumes of the reducing washing buffer
were applied. Thereafter, the GdmCl solution was replaced by 50 mM
sodium phosphate buffer (pH 8.0) comprising 100 mM NaCl, 10 mM
imidazole, and 5 mM TCEP to induce conformational refolding of the
matrix-bound SlyD fusion polypeptide. In order to avoid
reactivation of co-purifying proteases, a protease inhibitor
cocktail (Complete.RTM. EDTA-free, Roche) was added to the
refolding buffer. A total of 15 to 20 column volumes of refolding
buffer were applied in an overnight procedure. Thereafter, both
TCEP and the Complete.RTM. EDTA-free inhibitor cocktail were
removed by washing with 10 column volumes 50 mM sodium phosphate
buffer (pH 8.0) comprising 100 mM NaCl and 10 mM imidazole. In the
last washing step, the imidazole concentration was raised to 30 mM
(10 column volumes) in order to remove tenacious contaminants. The
refolded polypeptide was then eluted by applying 250 mM imidazole
in the same buffer. Protein-containing fractions were assessed for
purity by Tricine-SDS-PAGE (Schaegger, H. and von Jagow, G., Anal.
Biochem. 166 (1987) 368-379). Subsequently, the protein was
subjected to size-exclusion-chromatography (Superdex.TM. HiLoad,
Amersham Pharmacia) using potassium phosphate as the buffer system
(50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 0.5 mM
EDTA). Finally, the protein-containing fractions were pooled and
concentrated in an Amicon cell (YM10) to a concentration of
.about.5 mg/ml. Exemplarily SDS-PAGE analysis of Ni-NTA
purification of TtSlyD-FKBP-Her3 is shown in FIG. 3 and SEC elution
profile of a Ni-NTA purified fraction of Thermus thermophilus
SlyD-FKBP-Her-3 is shown in FIG. 4. The Thermus thermophilus SlyD
(TtSlyD)-Her-3 fusion polypeptide could be purified successfully as
a soluble and stable polypeptide in its monomeric form. The final
yield was quantified at 16.4 mg purified protein from fraction 12
and 13.
Table 2: Summary of the amino acid sequences of the developed
SlyD-based epitope scaffolds (which carry the HER3 dimerization
domain fragment ( -Hairpin of HER3 (SEQ ID NO: 1)) as insert or the
HER4 dimerization domain fragment ( -Hairpin of HER4 (SEQ ID NO:
2)) as insert).
TtSlyD-FKBP-Her3, TtSlyDcas-Her3, TtSlyDcys-Her3, Thermococcus
gammatolerans TgSlyDser-Her3 and TgSlyDcys-Her3 carry the Her3
dimerization domain fragment ( -Hairpin of HER3 (SEQ ID NO: 1)) as
insert and were used as immunogens and as positive controls in
ELISA screening.
TtSlyD-Wildtype, TtSlyDcas, TgSlyD.DELTA.IF were used as negative
controls in the ELISA screening (without the Her3 dimerization
domain fragment ( -Hairpin of HER3 (SEQ ID NO: 1)) or the Her4
dimerization domain fragment ( -Hairpin of HER4 (SEQ ID NO: 2)) as
insert).
TtSlyDcas-Her4, TtSlyDcys-Her4, TgSlyDser-Her4 and TgSlyDcys-Her4
(which carry the Her4 dimerization domain fragment ( -Hairpin of
HER4 (SEQ ID NO: 2)) as insert) were used in the ELISA screening to
check the developed clones for HER4 crossreactivity.
As the epitope scaffolds are expressed in E. coli the N-terminal
methionine residue can be present or not. (Nt=N-terminal;
Ct=C-terminal)
TABLE-US-00014 TABLE 2 TtSlyD- Nt- FKBP-
MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLE Her3
EALEGREEGEAFQAHVPAEKAYGAGSPQPLVYNKLTFQLEPNP
HTKGSSGKDLDFQVEVVKVREATPEELLHGHAHG GGSRKHHHHH HHH-Ct TtSlyD- Nt-
Wild- MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGL type
EEALEGREEGEAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDA
EVVPGAQFYAQDMEGNPMPLTVVAVEGEEVTVDFNHPLAGKD
LDFQVEVVKVREATPEELLHGHAHGGGSRKHHHHHHHH-Ct TtSly Nt- Dcas
MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLE
EALEGREEGEAFQAHVPAEKAYGAGSGSSGKDLDFQVEVVKV
REATPEELLHGHAHGGGSRKHHHHHHHH-Ct TgSly Nt- D.DELTA.I F
MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREY
SPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGATG
HPGIIPPHATAIFEIEVVEIKKAGEALEHHHHHHLEHHHHHH- Ct TtSly Nt- Dcas-
MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLE Her3
EALEGREEGEAFQAHVPAEKAYGAGSPQPLVYNKLTFQLEPNP
HTKGSSGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHH HHHHH-Ct TtSly Nt- Dcys-
MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGL Her3
EEALEGREEGEAFQAHVPAEKAYGPCGPQPLVYNKLTFQLEPN
PHTGCGKDLDFQVEVVKVREATPEELLHGHAHGGGSHHHHHH HH-Ct TgSly Nt- Dser-
MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREY Her3
SPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYGMPS
GPQPLVYNKLTFQLEPNPHTGSAGKTAIFEIEVVEIKKAGEAG GGSRKHHHHHHHH-Ct TgSly
Nt- Dcys- MRGSKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEE Her3
REYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYG
MPCGPQPLVYNKLTFQLEPNPHTGCAGKTAIFEIEVVEIKKAG EAGGGSHHHHHHHH-Ct TtSly
Nt- Dcas- MRSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLE Her4
EALEGREEGEAFQAHVPAEKAYGAGSPQTFVYNPTTFQLEHNF
NAKGSSGKDLDFQVEVVKVREATPEELLHGHAHGGGSRKHHH HHHHH-Ct TtSly Nt- Dcys-
MRGSKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGL Her4
EEALEGREEGEAFQAHVPAEKAYGPCGPQTFVYNPTTFQLEHN
FNAGCGKDLDFQVEVVKVREATPEELLHGHAHGGGSHHHHHH HH-Ct TgSly Nt- Dser-
MKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEEREY Her4
SPIGVTVGAGEIIPGIEEALLGMELGEKKEVVV
PPEKGYGMPSGPQTFVYNPTTFQLEHNFNAGSAGKTAIFEIEV
VEIKKAGEAGGGSRKHHHHHHHH-Ct TgSly Nt- Dcys-
MRGSKVERGDFVLFNYVGRYENGEVFDTSYESVAREQGIFVEE Her4
REYSPIGVTVGAGEIIPGIEEALLGMELGEKKEVVVPPEKGYG
MPCGPQTFVYNPTTFQLEHNFNAGCAGKTAIFEIEVVEIKKAG EAGGGSHHHHHHHH-Ct
Example 2
a) Immunisation and Selection of HER3 Antibodies
For the generation of antibodies against the -hairpin of HER3 and
HER4, Balb/C, NMRI or SJL mice were immunized with different
antigens. As antigens the following proteins were used: full length
Her3 ECD, or the epitope scaffold proteins TtSlyD-FKBP12-Her3,
TtSlyDcys-Her3, TtSlyDcas-Her3, TgSlyDcys-Her3 and TgSlyDser-Her3.
The TtSlyD-FKBP12-Her3 variant represents the first generation
epitope scaffold, used for generation of Her3 dimerization domain
specific antibodies. Although the general principal of using SlyD
variants as epitope scaffolds could already be demonstrated using
the first generation SlyD-FKBP12 scaffold, improved variants of the
scaffold with higher stability were developed. These SlyD variants
are derived from Thermos thermophilus and Thermococcus
gammatolerans.
All mice were subjected to 3 immunizations at the time points 0, 6
and 10 weeks after start of the immunization campaign. At each time
point each mouse was immunized with 100 .mu.g endotoxin free
immunogen dissolved in 100 .mu.l PBS. For the first immunization
the immunogen was mixed with 100 .mu.l CFA. For the second and
third immunization the immunogen was mixed with IFA. The first and
the third immunization were applied via the intraperitoneal route,
the second immunization was applied subcutaneously. 2 and 3 days
prior to the preparation of spleenocyte for antibody development
using hybridoma technology, the mice were subjected to intravenous
booster immunizations with 12.5 .mu.g immunogen in 100 .mu.l PBS
and without adjuvant.
Titer Analysis
For the determination of serum titers against the respective
immunogen and against the screening proteins a small amount of
serum of each mouse was collected in week 11 after start of the
immunization campaign. For the ELISA the immunogen or the screening
scaffold proteins were immobilized on the plate surface. Her3 ECD
was immobilized at a concentration of 1 .mu.g/ml and the scaffold
proteins TtSlyD-FKBP12-Her3, TtSlyD-FKBP12, TtSlyDcys-Her3,
TtSlyDcas-Her3, TtSlyDcas, TgSlyDcys-Her3, TgSlyDser-Her3 and
TgSlyD.DELTA.IF were used at a concentration of 0.5 .mu.g/ml. The
scaffold proteins TtSlyDcas and TgSlyD.DELTA.IF were used as
negative controls. The sera from each mouse were diluted in PBS
with 1% BSA and the dilutions were added to the plates. The sera
were tested at dilutions 1:300, 1:900, 1:2700, 1:8100, 1:24300,
1:72900, 1:218700 and 1:656100. Bound antibody was detected with a
HRP-labeled F(ab').sub.2 goat anti-mouse Fc.gamma. (Dianova) and
ABTS (Roche) as a substrate.
Even on the level of serum titration it was already obvious that
immunized mice developed antibodies against the Her3 -hairpin
domain. In mice immunized with Her3 ECD this can be shown by
titration against one of the scaffold proteins containing the
dimerization -hairpin loop. The strongly reduced signal can be
explained by the fact, that the majority of antibodies raised by
immunization with Her3 ECD are targeting other parts within the ECD
and only a small fraction is binding to the dimerization -hairpin
domain. In mice immunized with Her3 dimerization loop containing
scaffolds the fraction of antibodies targeting the loop can be
shown by titration against Her3 ECD (positive control) and
titration against an control scaffold without Her3 insertion
(negative control).
b) Antibody Development and ELISA Screening/Selection
The use of the here described epitope scaffold technology offers in
principal two strategies for the development of antibodies
targeting the Her3 dimerization domain ( -Hairpins of HER3 (SEQ ID
NO: 1)). One strategy is to immunize with the full length Her3 ECD
and to use the scaffolds to screen for the dimerization domain
specific antibodies. The other strategy is the direct use of the
scaffold for immunization and to use the Her3 ECD, a scaffold with
another backbone or a scaffold without insertion for counter
screening. Antibodies were developed with hybridoma technology by
fusing primary B-cells with P3X63Ag8.653 myeloma cells. 2 days
after the final booster immunization, immunized mice were
sacrificed and spleen cell populations were prepared. The
spleenocytes were fused with P3X63Ag8.653 by using the PEG fusion
technology. The cellular batch culture from the fusion was
incubated overnight at 37.degree. C. under 5% CO.sub.2. The
following day the cellular batch containing fused cells was
centrifuged for 10 min at 400 g. Thereafter, the cells were
suspended in hybridoma selection media supplemented with 0.1.times.
azaserine-hypoxanthine (Sigma) and were seeded at a concentration
of 2.5.times.10.sup.4 cells per well in 96 well plates. The plates
were cultured for at least 1 week at 37.degree. C. under 5%
CO.sub.2. 3 days prior to ELISA analysis the selection media was
changed.
Primary culture supernatants were tested in ELISA against Her3 ECD
and various scaffold proteins. The testing against the scaffold
proteins was done to demonstrate that the selected clones are
binding to the dimerization domain -hairpin of native Her3 ECD. The
testing against the control scaffolds TtSlyDcas and TgSlyD.DELTA.IF
was done to show that the selected clones are binding the inserted
Her3 derived sequence and not the scaffold backbone. To check for
cross reactivity the resulting clones were tested against the full
length ECDs of the other members of the Her family namely, Her1,
Her2 and Her4. As shown all selected clones are highly specific for
Her3 and a highly specific cross reactivity to HER4 could be
detected, while no cross reactivity to other members of the Her
family were detected. For the ELISA the screening an antigen down
format was used. Her3 ECD was immobilized at a concentration of 1
.mu.g/ml and the scaffold proteins TtSlyD-FKBP12-Her3,
TtSlyD-FKBP12, TtSlyDcys-Her3, TtSlyDcas-Her3, TtSlyDcas,
TgSlyDcys-Her3, TgSlyDser-Her3 and TgSlyD.DELTA.IF were immobilized
at a concentration of 0.5 .mu.g/ml. Hybridoma Supernatant was added
to the plates and incubated for 1 h at room temperature. Bound
antibody was detected with a HRP-labeled F(ab').sub.2 goat
anti-mouse Fc.gamma. (Dianova) and ABTS (Roche) was used as a
HRP-substrate.
TABLE-US-00015 TABLE 3 Evaluation of the selected clones by ELISA.
The clones were tested against the scaffold proteins
TtSlyDcas-Her3, TtSlyDcys-Her3, TgSlyDser-Her3 and TgSlyDcys-Her3
and the full length Her3 ECD to verify their Her3 dimerization
domain insert (.beta.-Hairpin of HER3 (SEQ ID NO: 1)) specificity.
As negative controls the scaffold proteins TtSlyDcas and
TgSlyD.DELTA.IF were used. Additionally, clones were tested against
full length ECDs of Her1, Her2, Her3 and Her4 to verify potential
cross reactivity. Clones show binding to full length Her3 ECD and
are cross reactive against full length Her4 ECD. TtSlyD- TgSlyD-
cas- cys- ser- cys- Her1 Her2 Her3 Her4 Clone cas Her3 Her3
.DELTA.IF Her3 Her3 ECD ECD ECD ECD M-05-74 0.023 3.133 3.150 0.020
3.159 3.159 0.018 0.020 3.152 3.170 M-15-02 0.040 1.763 1.522 0.040
1.980 1.785 0.024 0.025 3.153 3.192 M-15-03 0.045 1.772 1.850 0.039
1.628 1.461 0.020 0.024 3.171 3.234 M-15-04 0.040 1.847 1.457 0.033
1.833 1.500 0.067 0.064 3.175 3.186 M-15-05 0.041 1.443 1.482 0.046
1.886 1.485 0.020 0.021 3.156 3.216 M-15-08 0.041 1.569 1.707 0.040
1.746 1.532 0.019 0.023 3.195 3.181 M-15-09 0.057 1.870 1.929 0.076
1.799 1.640 0.024 0.037 3.234 3.200 M-15-11 0.044 1.714 1.636 0.056
2.005 1.693 0.029 0.031 3.103 3.218 M-16-01 0.039 1.653 1.793 0.037
1.860 1.637 0.024 0.032 3.184 3.212
c) Immunohistochemistry
All selected clones were tested for reactivity and specificity in
IHC. Therefore HEK293 cells were transiently transfected with
plasmids coding for full length HER1, HER2, HER3 or HER4,
respectively. 2 days after transfection the different cell lines
now expressing HER1, HER2, HER3 or HER4 were harvested,
subsequently fixed in formalin and embedded in Agarose for
generation of IHC controls. After an additional fixation in
formalin overnight the Agarose blocks were embedded in paraffin.
Untransfected HEK293 cells were used as negative controls and
treated accordingly to the transfected cells. After paraffin
embedding 3 .mu.m thin sections were prepared using a microtome.
The sections were mounted on glass microscopy slides and dried for
2 h. All further steps of the immunohistochemical staining
procedure were carried out using a Ventana Benchmark.RTM. XT. The
slides were dewaxed and antigen retrieval was performed by applying
heat for 1 hour. For antigen retrieval the Ventana buffer CC1 was
used. The antibodies were used at a concentration of 1 .mu.g/ml.
For the detection of bound antibody the Ventana UltraView.TM.
detection kit was used. Results are shown in FIG. 5. All three
clones showed binding to HER3 and cross reactivity against HER4. No
cross reactivity against HER1 and HER2 was detectable.
d) DNA Sequencing of Selected Anti-Her3 Hybridoma
To obtain the DNA sequences of the selected hybridoma clones a 5'
Race PCR was conducted. For the RT-PCR total RNA was prepared from
5.times.10.sup.6 cells by using a total RNA purification kit
(Qiagen). The reverse transcription and the PCR were conducted
using a 5'prime RACE PCR kit (Roche). The resulting PCR fragments
from heavy and light chain were purified by gel electrophoresis and
subsequent gel purification. The PCR fragments were cloned using
the Topo.RTM. Zero-Blunt.RTM. cloning kit (Invitrogen) and
transformed into competent cells. Several clones from each
hybridoma were submitted for sequencing to obtain a consensus
sequences for the selected clones. M-05-74 M-15-02 M-15-04 were
submitted for sequencing which resulted in identical VH and VL
sequences for all 3 clones. M-15-03, M-15-05, M-15-08, M-15-09,
M-15-11, M-16-01 were sequenced analogously and also resulted in
identical VH and VL sequences for all clones.
e) Time Dependent Internalization Analyses of M-05-74 Via FACS
Binding and internalization of HER3 by the selected clone M-05-74
to HER3 was analyzed in FACS using the HER3 expressing tumor cell
line T47D. 5.times.10.sup.5 cells were treated with 50 ng
Recombinant Human Heregulin fragment (HRG) (SEQ ID NO: 11). The
fragment including amino acid of SEQ ID NO: 11 was cloned in
pCDNA.1 vector (Invitrogen). The HRG fragment was expressed in
FreeStyle.TM. 293-F cells according to the protocol described by
Invitrogen. (FreeStyle.TM. 293 Expression system Catalog no.
K9000-01). Purified HRG fragment was solved in 20 mM Histidin, 140
mM NaCl; pH6.0 and stored by -80 C.
Untreated (-) cells were used as negative controls. Shortly after
Heregulin induced activation, 1 .mu.g of M-05-74 was added to the
cells. The cells were incubated for 0, 5, 15, 30, 45, 60, 75, 90,
105, 120, 180 or 240 min at 37.degree. C. After incubation the
cells were immediately put on ice. The cells were washed with 3 ml
FACS buffer once and then stained for 30 minutes with 1 .mu.g of a
R-Phycoerythrin Goat Anti-Mouse IgG (H+L) secondary antibody. Flow
cytometry was carried out using a FACSCanto.TM. flow cytometer (BD
Biosciences). Results are FACS analysis of M-05-74 induced, time
dependent HER3 receptor internalization in T47D cells. M-05-74
shows binding to the expressed HER3 ECD, with or without
supplemented recombinant human Heregulin fragment (HRG). M-05-74
leads to Her3 receptor internalization over a 4 h time period.
Results are shown in FIG. 6. The isotype control is indicated as a
constant horizontal black bar. M-05-74 shows binding to the
expressed Her3 ECD, with or without Human Heregulin fragment (-)
and (+HRG). M-05-74 leads to Her3 receptor internalization over a 4
h time period. The isotype control is indicated as a constant
horizontal black bar. In the presence of HRG the antibody induced
internalization of HER3 was faster (e.g after 1 h, at least 25%
more HER3 were internalized in the presence of HRG (+HRG) when
compared to the value in the absence of HRG (-).
Example 3
a) Kinetic Screening/Binding Properties of HER3 Antibodies
The kinetic screening was performed according to Schraeml et al.
(Schraml, M. and M. Biehl, Methods Mol Biol 901 (2012) 171-181) on
a Biacore.TM. 4000 instrument, mounted with a Biacore.TM. CM5
sensor. In all assay the test antibodies were captured. The system
was under the control of the software version V1.1. The instrument
buffer was HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA,
0.05% (w/v) P20). The system operated at 25.degree. C. 30 .mu.g/ml
Rabbit polyclonal antibody (RAM IgG, (Rabbit anti Mouse IgG with Fc
gamma specificity) GE Healthcare) in 10 mM sodium acetate buffer
(pH 4.5) was immobilized using EDC/NHS chemistry according to the
manufacturer's instructions on the spots 1, 2, 4 and 5 in the flow
cells 1, 2, 3 and 4. The sensor was saturated using 1M
ethanolamine. In each flow cell, referenced signals were calculated
using spots 1-2 and spots 5-4, spot 3 served as a blanc control.
The antigen (human recombinant Her-3 ECD (68 kDa), and recombinant
Thermus thermophilus SlyD FKBP-Her3 (15 kDa) comprising the
-hairpin peptide of HER3 (SEQ ID NO:1)) was diluted at 150 nM in
instrument buffer supplemented with 1 mg/ml
CMD(Carboxymethyldextran, Sigma). to suppress unspecific binding.
Prior to their application the hybridoma culture supernatants were
diluted 1:5 in instrument buffer. The diluted mixtures were
injected at a flow rate of 30 .mu.l/min for 2 min. The antibody
capture level (CL) in response units was monitored. Immediately
thereafter the respective antigen was injected at a flow rate of 30
.mu.l/min for 3 min association time. Thereafter, the
antibody-antigen complex dissociation signal was recorded for 5
min. The sensor was regenerated by injecting a 10 mM glycine-HCl
solution (pH 1.7) for 2 min at a flow rate of 30 .mu.l/min. The
recorded signal shortly before the end of the injection of the
antigen was denoted as binding late (BL) in response units. The
recorded signal shortly before the end of the recording of the
dissociation is denoted as stability late (SL) in response units.
The dissociation rate constants were determined calculated The
antibody-antigen complex stability in minutes was calculated with
the following formula: ln(2)/60*kd. The Molar Ratio was calculated
with the formula: MW (antibody)/MW (antigen) *BL (antigen)/CL
(antibody).
Binding Late (BL) represents the response units at the end of the
analyte injection. The amount of antibody captured as a ligand on
the sensor surface is measured as Capture Level (CL) in response
units. Together with the information of the molecular weights of
the tested analytes, the antibody and the analyte in solution, the
Molar Ratio can be calculated. In case the sensor was configurated
with a suitable amount of antibody ligand capture level, each
antibody should be able to functionally bind at least to one
analyte in solution, which is represented by a Molar Ratio of
MR=1.0. Then, the Molar Ratio is also an indicator for the valence
mode of analyte binding. The maximum valence can be MR=2 for an
antibody binding two analytes, one with each Fab valence. In case
of steric limitations or a dysfunctional analyte binding, the Molar
Ratio can indicate understoichiometric binding, like it is the case
when the Her-3 ECD is being bound in its "closed" conformation. The
maximum assay deviation in the determination of the Molar Ratio is
MR=0.2.
Screening/Selection of Anti-HER3/HER4 Antibody M-05-74:
In one experiment, the kinetic screening was driven with hybridoma
primary cultures from different fusions, which were obtained from
an immunization of mice with human recombinant Her-3 ECD. The aim
was to select cultures with binding specificity for the Her-3
heterodimerization domain -hairpin peptide (SEQ ID NO:1). As
antigens in solution human recombinant Her-3 ECD (68 kDa), and
recombinant Thermus thermophilus SlyD FKBP-Her3 (15 kDa) comprising
the -hairpin peptide of HER3 (SEQ ID NO:1) were used. A positive
hit was classified as a primary culture supernatant with binding
activity versus both antigens.
The Table 4 exemplarily shows primary culture supernatants, from
which M-05-74 fulfills these requirements, indicating epitope
specificity for the -hairpin of HER3. Therefore this is a suitable
method of screening of anti-HER3 antibodies which bind to the Her-3
hairpin of SEQ ID NO:1.
TABLE-US-00016 TABLE 4 Exemplary results obtained from a kinetic
screening experiment with a set of hybridoma primary cultures from
fusions, wherein antibody M-05-74 was identified as binding to both
HER3 ECD and the .beta.-hairpin of HER3 (SEQ ID NO: 1) within the
thermo SlyD-Her3 construct. binding stability late BL late SL kd
t/2 diss T CL MR Ligand Analyte [RU] [RU] [1/s] [min] [.degree. C.]
[RU] [--] M-04-06 human-Her3-ECD 17 16 4.13E-04 28 25 134 0.3
M-04-06 thermo SlyD-Her3 -4 -4 n.d. n.d. 25 134 -0.3 M-04-140
human-Her3-ECD -1 1 n.d. n.d. 25 110 0.0 M-04-140 thermo SlyD-Her3
-6 -5 n.d. n.d. 25 112 -0.5 M-05-20 human-Her3-ECD 32 33 4.98E-05
232 25 623 0.1 M-05-20 thermo SlyD-Her3 -9 -6 n.d. n.d. 25 625 -0.1
M-05-30 human-Her3-ECD 122 123 3.74E-05 309 25 521 0.5 M-05-30
thermo SlyD-Her3 -3 -2 n.d. n.d. 25 525 -0.1 M-05-44 human-Her3-ECD
55 55 3.42E-05 337 25 373 0.3 M-05-44 thermo SlyD-Her3 -7 -6 n.d.
n.d. 25 369 -0.2 M-05-74 human-Her3-ECD 75 79 <1.00E-05 >1155
25 318 0.5 M-05-74 thermo SlyD-Her3 33 32 1.20E-04 96 25 315 1.1
M-05-82 human-Her3-ECD 0 1 n.d. n.d. 25 205 0.0 M-05-82 thermo
SlyD-Her3 -4 -5 n.d. n.d. 25 204 -0.2
It has been found that M-05-74 shows a reduced Molar Ratio in its
binding to the human Her-3 ECD analyte (MR=0.5), whereas in its
binding to analyte Thermus thermophilus SlyD FKBP-Her3 comprising
the -hairpin HER3 (SEQ ID NO:1) M-05-74 shows an improved Molar
Ratio (MR=1.1), indicating a functional, stoichiometric 1:1 binding
with improved epitope accessibility (compared to human Her-3
ECD).
b) Kinetics of HER3 Antibodies M-05-74, M-205 and M-208 Kinetics to
Investigate the Mode of Action of M-05-74 in the Absence and
Presence of Heregulin (HRG)
In its equilibrium state, the Her-3 ECD is in its "closed
confirmation", which does mean, the heterodimerization Her-3
beta-hairpin motive is tethered via non-covalent interactions to
the Her-3 ECD domain IV (see FIGS. 1c and d). It is supposed, that
the "closed" Her-3 conformation can be opened via the binding of
the ligand heregulin at a specific Her-3 heregulin binding site.
This takes place at the Her-3 interface formed by the Her-3 ECD
domains I and domain III. By this interaction it is believed, that
the Her-3 receptor is activated and transferred into its "open
conformation" (see FIGS. 1b and e). When this occurs, the Her-3
beta-hairpin is accessible for the described antibodies. This mode
of action can be simulated in vitro by a Biacore.TM.
experiment.
A Biacore.TM. T100 instrument (GE Healthcare) was used to
kinetically assess the monoclonal antibodies for their behavior to
the heregulin-activated Her-3 Extracellular Domain (Her3_ECD). A
CM5 series sensor was mounted into the system and was normalized in
HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%
w/v Tween 20) according to the manufacturer's instructions. The
sample buffer was the system buffer supplemented with 1 mg/ml CMD
(Carboxymethyldextran, Sigma #86524). The system operated at
25.degree. C. 6500 RU RAM-Fc.gamma. (relative units of
Fc.gamma.-fragment RamIgG, GE Healthcare) were immobilized
according to the manufacturer's instructions using EDC/NHS
chemistry on all four flow cells. The sensor was deactivated using
1M ethanolamine.
The binding activity of the respective antibody against the
analytes was kinetically tested. Antibodies were captured at 35 nM
concentration by a 1 min injection at 5 .mu.l/min. The flow rate
was set to 100 .mu.l/min.
The analytes in solution tested were human Heregulin fragment (HRG)
(SEQ ID NO:11), a 44 kDa homodimeric protein (prepared according to
Example 2e), human recombinant HER2 ECD (SEQ ID NO:10) (69.6 kDa),
human recombinant HER3 ECD (SEQ ID NO:4) (68 kDa), human
recombinant HER4 ECD (SEQ ID NO:6), and 100 nM of the Her-3 ECD and
the Her-4 ECD each incubated with a 5-fold molar excess of
Heregulin for 60 min at room temperature resulting in HER3 ECD-HRG
complex and HER4 ECD-HRG complex (Addition of MWs for
complexes).
Analytes in solution were injected at different concentration steps
of 0 nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM and 90 nM for 3.5 min.
The dissociation was monitored for 15 min. Where possible, kinetic
signatures were evaluated according to a Langmuir fit.
TABLE-US-00017 TABLE 5a SPR-resolved kinetic data of M-05-74
(=M-074), M-205 and M-208 CL Analyte T k.sub.a k.sub.d K.sub.D
K.sub.D BL Chi.sup.2 Antibody RU in solution .degree. C. 1/Ms 1/s M
nM RU MR RU.sup.2 M-074 535 HRG 25 n.d. n.d. n.d. n.d. n.d. n.d.
n.d. M-074 530 HER2_ECD 25 n.d. n.d. n.d. n.d. n.d. n.d. n.d. M-074
648 HER3-ECD 25 1.3E+04 2.8E-05 2.2E-09 2 70 0.2 0.1 M-074 712
HER4-ECD 25 6.7E+03 1.0E-03 1.5E-07 150 27 0.1 0.1 M-074 546 HER3-
25 6.3E+04 2.7E-04 4.2E-09 4 160 0.6 2.3 ECD-HRG M-074 719 HER4- 25
1.6E+05 8.3E-04 5.2E-09 5 349 0.6 0.0 ECD-HRG M-205 591 HRG 25 n.d.
n.d. n.d. n.d. n.d. n.d. n.d. M-205 588 HER2_ECD 25 n.d. n.d. n.d.
n.d. n.d. n.d. n.d. M-205 605 HER3-ECD 25 4.9E+04 1.0E-04 2.0E-09 2
235 1.0 1.3 M-205 597 HER3- 25 3.7E+04 1.2E-04 3.2E-09 3 164 0.4
0.3 ECD-HRG M-208 777 HRG 25 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
M-208 771 HER2_ECD 25 n.d. n.d. n.d. n.d. n.d. n.d. n.d. M-208 822
HER3-ECD 25 5.8E+04 5.3E-05 9.1E-10 1 367 1.0 9.4 M-208 795 HER3-
25 5.0E+04 1.4E-04 2.8E-09 3 390 1.1 17.6 ECD-HRG MR = Molar Ratio,
BL = Binding Late, CL = Capture Level; n.d. = not detectable = no
bindingThe Molar Ratio was calculated with the formula: MW
(antibody)/MW(antigen) *BL (antigen)/CL (antibody).
The antibody M-205 is a murine monoclonal antibody with binding
activity versus an epitope nearby the Her-3 ECD Heregulin binding
site (described as Mab205.10.2 in WO2011/076683). M-205 competes
with Heregulin around its binding site on the Her-3 ECD.
The antibody M-208 is a murine monoclonal antibody with binding
activity versus the Her-3 ECD domain IV. M-208 binds to the Her-3
ECD independently of the Her-3 ECD conformational state.
M-05-74 (=M-074 in Table 5) binds to the Her-3 ECD in its active
"open" conformation (on the presence of ligand (e.g. heregulin HRG)
with improved kinetics, due to a better accessibility of the Her-3
hairpin in its "open" conformation. The MR is at least two fold
higher.
No antibody binding (n.d.) was observed versus the negative control
analytes Heregulin beta (HRG) and the extracellular HER-2 domain
(HER2_ECD). The tested antibodies showed all binding to the
Her3-ECD (HER3_ECD), but with strongly differing BL values.
M-05-74 binds to the Her-3 ECD in its "closed" conformation with
slower association rate constant k.sub.a=1.3E+04 1/Ms and smaller
BL (70 RU) than when compared to the clones M-205 with faster
k.sub.a=4.9 E+04 1/Ms and high signal amplitude at BL (235 RU) and
M-208 with faster k.sub.a=5.8E+04 1/Ms and also high signal
amplitude at BL (367RU). This implicates on the stoichiometry of
the binding (MR), where M-205 (MR=1.0) and M-208 (MR=1.0) both show
a functional 1:1 binding for the HER3-ECD, whereas M-05-74 shows a
non-functional binding (MR=0.2). Here it is supposed, that this
interaction of M-05-74 versus the Her-3 ECD is residual binding of
a portion of structurally handicapped Her-3 ECD analyte. This is
also supposed for the interaction of M-05-74 versus the Her-4 ECD,
which also shows a non-functional binding with BL (27 RU) and
(MR=0.1).
A surprising result is the more than 4-fold increase (nearly 5
fold) of the M-05-74 association rate constant k.sub.a from the
"closed" Her-3 ECD to the "open" Her-3 ECD/Heregulin complex; from
k.sub.a=1.3E+04 1/Ms (Her3 ECD) to k.sub.a=6.3E+04 1/Ms
(Her3-ECD-HRG). So M-05-74 binds to HER3-ECD with a ratio of the
association constant (K.sub.a) in presence of Heregulin (Ka
(+Heregulin)) and absence of Heregulin (Ka (-Heregulin)) of 4.0 or
higher (Ka (+Heregulin))/(Ka (-Heregulin)=ka (Her3-ECD-HRG)/ka
(Her3-ECD)=6.3E+04 [1/Ms]/1.3E+04 [1/Ms])=4.85)). Thereby the Molar
Ratio improves 3-fold, indicating now a 1:1 interaction of M-05-74
with the Her-3 ECD Heregulin complex. Thus binds M-05-74 to
HER3-ECD with a ratio of the Molar Ratio MR of binding in presence
of Heregulin (MR (+Heregulin)) and in absence of Heregulin (MR
(-Heregulin)) of 3.0 (MR (+Heregulin))/(MR
(-Heregulin)=0.6/0.2=3).
This is also valid for the Her-4 ECD/Heregulin complex, where the
Molar Ratio improves 6-fold, indicating a 1:1 interaction of
M-05-74 with the Her-4 ECD Heregulin complex. Thus binds M-05-74 to
HER4-ECD with a ratio of the Molar Ratio MR of binding in presence
of Heregulin (MR (+Heregulin)) and in absence of Heregulin (MR
(-Heregulin)) of 3.0 (MR (+Heregulin))/(MR (-Heregulin)=0.6/0.1=6).
And furthermore surprisingly the M-05-74 association rate constant
ka increases from the "closed" Her-4 ECD to the "open" Her-4
ECD/Heregulin complex from ka=6.7E+03 1/Ms (Her3_ECD) to ka=1.6E+05
more than 20-fold. So M-05-74 binds to HER4-ECD with a ratio of the
association constant (Ka) in presence of Heregulin (Ka
(+Heregulin)) and absence of Heregulin (Ka (-Heregulin)) of 20.0 or
higher (Ka (+Heregulin))/(Ka (-Heregulin)=ka (Her4-ECD-HRG)/ka
(Her4-ECD)=6.7E+04 [1/Ms]/1.6E+05 [1/Ms])=23.88)).
As expected, the Heregulin displacer M-205, reduces its BLvalue and
the Molar Ratio. The Molar Ratio is decreased 2.5-fold, from a
fully functional 1:1 interaction with MR=1.0 (Her3-ECD) with 235 RU
at BL into a less functional MR=0.4 (Her3-ECD-HRG) with 164 RU at
BL. This indicates the loss in functionality due to the competing
presence of excess Heregulin.
The antibody M-208, which binds to the Her-3 ECD domain IV remains
completely unaffected by the presence of Heregulin. No significiant
change of the Molar Ratios MR could be detected.
The FIG. 7 shows the mode of binding of the anti-HER3/HER4 -hairpin
antibody M-05-74 to the Heregulin-activated Her-3 ECD complex.
M-05-74 (see plot 1) captures and prevents the Heregulin
dissociation from the complex. M-05-74 is a trap for Heregulin
("Heregulin-sink"). M-05-74 does not compete with Heregulin for a
binding site on the Her-3 ECD. For comparison M-08-11 (plot 2) is
shown; M-08-11 (VH and VL see SEQ ID NO: 51 and 52) is another HER3
-Hairpin binder with no HER4 ECD and HER4 -hairpin crossreactivity,
which binds to a different epitope than M-05-74.
Ia further experiment also HER1 ECD, T.T.SlyD-cysHer3 and
T.T.SlyD-cas without the HER3 -hairpin were included in the
measurement--results are shown in Table 5b, which substantially
reveals the same binding properties of M-05-74.
A Biacore.TM. T200 instrument (GE Healthcare) was mounted with a
CM5 series sensor. The sensor was normalized in HBS-ET buffer (10
mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w/v Tween 20)
according to the manufacturer's instructions. The sample buffer was
the system buffer supplemented with 1 mg/ml CMD
(Carboxymethyldextran, Sigma #86524). The system operated at
25.degree. C. 6500 RU RAM-Fc.gamma. (relative units of
Fc.gamma.-fragment RamIgG, GE Healthcare) were immobilized
according to the manufacturer's instructions using amine coupling
EDC/NHS chemistry on all four flow cells. The sensor was
deactivated using 1M ethanolamine. Monoclonal antibodies were
captured (CL, Capture Level) on the sensor surface by a 1 min
injection at 10 .mu.l/min. Concentration dependent kinetics were
measured. A concentration series of the analytes HER-1-ECD,
HER-2-ECD, HER-3-ECD, HER-4-ECD, T.T.SlyD-cysHer3 and T.T.SlyD-cas
were injected each at 0 nM, 1.1 nM, 3.3 nM, 2.times.10 nM, 30 nM
and 90 nM. Heregulin beta (HRG) was injected at 0 nM, 17 nM,
2.times.50 nM, 150 nM and 450 nM, 90 nM HER-3 ECD and 90 nM HER-4
ECD were preincubated for 2 hrs with a five-fold molar excess of
HRG beta and were injected at HER concentrations steps of 0 nM, 1.1
nM, 3.3 nM, 2.times.10 nM, 30 nM and 90 nM. All analytes were
injected for 5 min association time and 10 min dissociation time at
100 .mu.l/min flow rate. The sensor capture system was regenerated
by a 3 min injection at 10 .mu.l/min of 10 mM glycine pH 1.7. Where
possible kinetic data was evaluated using the Biacore.TM. T200
evaluation software. HER-3-ECD, HER-4-ECD and T.T.SlyD-cysHer3
kinetics were evaluated using a Langmuir fitting model.
HER-3-ECD-HRG and HER-4-ECD-HRG kinetics of M-5-74, were evaluated
according to a Langmuir fitting model.
TABLE-US-00018 TABLE 5b SPR-resolved kinetic data of M-05-74 CL
Analyte (Ab) k.sub.a kd K.sub.D RMax Chi.sup.2 T Antibody in
solution RU 1/Ms 1/s M RU MR RU.sup.2 .degree. C. M-5-74 HER1-ECD
288 n.d. n.d. n.d. 1 n.d. 0 25 HER2-ECD 287 n.d. n.d. n.d. 1 n.d. 0
HER3-ECD 289 9.6E+04 1.1E-04 1.1E-09 19 0.1 0.05 HER4-ECD 285
1.6E+04 8.2E-04 5.1E-08 13 0.1 0.01 HER3- 312 1.0E+05 2.9E-04
2.8E-09 195 0.8 2.2 ECD-HRG HER4- 301 9.9E+04 8.1E-04 8.1E-09 179
0.8 1.8 ECD-HRG HRG 301 n.d. n.d. n.d. 0 n.d. 0.0 T.T.SlyD- 486
3.0E+04 2.4E-04 7.8E-09 88 1.9 0.02 cysHer3 T.T.SlyD- 490 n.d. n.d.
n.d. 0.5 0.0 0.06 cas MR = Molar Ratio, BL = Binding Late, CL =
Capture Level; n.d. = not detectable = no bindingM-05-74 binds
HER-3-ECD-HRG and HER-4-ECD-HRG with 1:1 stoichiometry and inactive
HER-3-ECD and HER-4-ECD with 10:1 stoichiometry. M-05-74 binds
HER-3-ECD and HER-3-ECD-HRG with higher affinity than HER-4-ECD and
HER-4-ECD-HRG. M-05-74 does not interact with HER-1, HER-2 and HRG.
M-05-74 binds T.T.SlyD-cysHer3 with 1:2 stoichiometry and does not
interact with T.T.SlyD-cas.
Example 4
Epitope Mapping of Anti-HER3 Antibody M-05-74 and Mode of Action
Analysis
M-05-74 with a Unique Epitope ( -Hairpin of HER3 and HER4)
A Biacore.TM. 2000 (GE Healthcare) instrument was used to assess
the accessible epitopes clone culture supernatants for their
binding specificity. A CM5 sensor was mounted into the system and
was normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3
mM EDTA, 0.005% w/v Tween 20) according to the manufacturer's
instructions. The sample buffer was the system buffer supplemented
with 1 mg/ml CMD (Carboxymethyldextran, Sigma). The system operated
at 37.degree. C. 10000 RU RAM-Fc.gamma. (relative units of
Fc.gamma.-fragment Rabbit Anti-Mouse IgG/Jackson Laboratories) were
immobilized according to the manufacturer's instructions using
EDC/NHS chemistry on all four flow cells. The sensor was
deactivated using 1M ethanolamine.
At a flow rate of 10 .mu.l/min the primary antibody 50 nM anti-HER3
M-05-74 was captured for 1 min on all flow cells. The flow rate was
set to 30 .mu.l/min and an IgG blocking solution (50 .mu.g/ml IgG
(20:2:1 IgG1-Fc.gamma., IgG2a-Fc.gamma., IgG2b), Roche) was
injected for 5 minutes. The antigen Her-3 ECD was injected at 1.5
.mu.M for 3 min.
Afterwards, 100 nM of each anti-HER3 secondary antibodies (a)
M-05-74 b) 8B8 from WO97/35885 (named GT in the Figure) c) M-208
which binds to domainIV of HER3, and d) M-08-11; another HER3
-Hairpin binder with no HER4 ECD and HER4 -hairpin crossreactivity)
was injected for 3 minutes at 30 .mu.l/min. Acidic regeneration of
the sensor surface was achieved using three consecutive injections
of 10 mM Glycine pH 1.7 at 30 .mu.l/min for 60 sec.
The noise of the measurement is defined by the rebinding of the
secondary M-05-74 injection, which re-saturates the already
dissociated primary M-05-74. The experiment showed (see FIG. 8),
that M-208 and M-05-74 occupy distinct epitopes on the Her-3 ECD,
because the secondary M-208 signal completely saturates the Her-3
ECD in the presence of M-05-74. M-08-11 binding is completely
blocked by the presence of M-05-74. The M-08-11 secondary signal is
even below noise.
Nevertheless M-08-11 binds to a different epitope than M-05-74 as
M-08-11 does not bind to human HER4 ECD and HER4 -hairpin. (see
also below the exact epitope mapping data with the -hairpins of
HER3 and HER4). The 8B8 (=GT) secondary antibody produces a
significant signal in the presence of M-05-74, which is above
noise. Therefore the 8B8 (=GT) antibody binds another epitope than
M-05-74 and M-08-11.
M-05-74 with Unique Epitope and Mode of Actions
A Biacore.TM. B3000 instrument (GE Healthcare) was used to
kinetically assess the clone culture M-05-74 and the antibody 8B8
(from WO 97/35885, named GT in the Figures) to the "closed"
conformation of Her-3 ECD and the "open", Heregulin-activated Her-3
ECD. A CM5 series sensor was mounted into the system and was
normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM
EDTA, 0.005% w/v Tween 20) according to the manufacturer's
instructions. The sample buffer was the system buffer supplemented
with 1 mg/ml CMD (Carboxymethyldextran). The system operated at
25.degree. C. 10000 RU RAM-Fc.gamma. (relative units of
Fc.gamma.-fragment Rabbit Anti-Mouse IgG/Jackson Laboratories) were
immobilized according to the manufacturer's instructions using
EDC/NHS chemistry on all flow cells. The sensor was deactivated
using 1M ethanolamine. Analytes in solution were injected at 100
.mu.l/min at different concentration steps of 0 nM, 1.1 nM, 3.7 nM,
11.1 nM, 33.1 nM and 90 nM for 2 min. The dissociation was
monitored for 5 min. Acidic regeneration of the sensor surface was
achieved using three consecutive injections of 10 mM Glycine pH 1.7
at 30 .mu.l/min for 60 sec. Kinetic data were evaluated according
to a Langmuir fit.
TABLE-US-00019 TABLE 6 Langmuir kinetics of M-05-74 in comparison
to 8B8 (GT). 8B8 with lower antigen complex stability (t/2diss) and
less functionality (MR). CL Analyte in T ka t/2-diss BL Chi.sup.2
Antibody (RU) solution (.degree. C.) (1/Ms) (min) (RU) MR
(RU.sup.2) 8B8 339.3 ECD-HRG 25 3.21E+05 0.8 90 0.4 2.57 M-074
314.7 ECD-HRG 25 6.6E+04 18 199 0.8 0.773 8B8 347.3 Her-3 ECD 25
1.02E+05 5.3 13.1 0.1 0.12 M-074 318.2 Her-3 ECD 25 2.04E+04 28 36
0.2 0.122 8B8 476.1 ttSlyD-Her3 25 n.d. n.d. n.d. n.d. n.d. M-074
468 ttSlyD-Her3 25 8.75E+04 4.9 68.1 1.5 0.174 MR = Molar Ratio, BL
= Binding Late, CL = Capture Level
In the table above kinetic data of the antibody clone M-05-74 and
the antibody 8B8 are listed. M-05-74 binds to the
Heregulin-activated Her-3 ECD with high functionality MR=0.8.
M-05-74 and acts as Heregulin trap. (see also Biacore.TM. sensogram
Example 3b and FIG. 7).
The complex stability of the 8B8 antibody with t1/2 diss=0.8 min is
weak. 8B8 binds with an, MR=0.4. No separated dissociation phases
of the 8B8 antibody and the Heregulin dissociation can be
identified. Heregulin completely dissociates off in the same
timeframe and with the same velocity, like 8B8. 8B8 antibody does
not delay the heregulin dissociation.
M-05-74 functionally binds (MR=1.5) to the Thermus thermophilus
SlyD FKBP-Her3 comprising th HER3 -Hairpin of SEQ ID NO:1 with
KD=27 nM. Since the antibody 8B8 does not bind to the HER3 -Hairpin
comprising Thermus thermophilus SlyD FKBP-Her-3 fusion polypeptide
this antibody targets another epitope than M-05-74.
FIG. 9 is an overlay plot of the Biacore.TM. sensogramms of
anti-HER3/HER4 antibody M-05-74, anti-HER3 antibody M-08-11 and
anti-HER3 antibody 8B8 (from WO97/35885) showing the different
binding modes of actions. Anti-HER3/HER4 antibody M-05-74 traps the
Heregulin-activated Her-3 ECD (1) with t1/2 diss=18 min and acts
Heregulin-sink. Anti-HER3 antibody M-08-11 HER3 ( -Hairpin binder
with no HER4 ECD and HER4 -hairpin crossreactivity) delays the
Heregulin dissociation (2) and produces a complex two-state
kinetic. 8B8 antibody (3) is does not trap Heregulin and also not
delays the Heregulin dissociation from the Her-3 ECD/Heregulin
complex. Since it is a perfect Langmuir interaction, the
Heregulin/Her-3 ECD complex quickly and completely dissociates as
intact complex from the 8B8 antibody.
In FIG. 10 a scheme of these binding modes of action is shown: 1:
M-08-11 binds to the Heregulin activated Her-3 ECD and induces a
delayed Heregulin dissociation, whereby M-08-11 stays in the Her-3
ECD receptor complex. 2: M-05-74 binds to the Heregulin activated
Her-3 ECD. Heregulin is trapped in the complex and the antibody
stays in the complex. 3: 8B8 binds the Heregulin activated Her-3
ECD. The whole complex dissociates from the antibody.
Peptide-Based 2D Epitope Mapping
In another embodiment a peptide-based epitope mapping experiment
was done to characterize the Her-3 ECD epitopes by using the
CelluSpots.TM. Synthesis and Epitope Mapping technology. Epitope
mappings were carried out by means of a library of overlapping,
immobilized peptide fragments (length: 15 amino acids)
corresponding to the sequences of human Her-1 ECD, Her-2 ECD,Her-3
ECD and Her-4 ECD peptide hairpins. In FIG. 11, the strategy of the
epitope mapping and alanine-scan approach is shown. The peptide
hairpin sequences ( -hairpin) of HER1(EGFR) ECD, HER2 ECD,HER3 ECD
and HER4 ECDincluding their structural embeddings (structural) were
investigated. Cysteins were replaced by serines. For antibody
selection of the antibodies via binding to such -hairpins, the
-hairpins of HER3 and HER4 are defined by SEQ ID NO:1 and SEQ ID
NO:2.
Each peptide synthesized was shifted by one amino acid, i.e. it had
14 amino acids overlap with the previous and the following peptide,
respectively. For preparation of the peptide arrays the Intavis
CelluSpots.TM. technology was employed. In this approach, peptides
are synthesized with an automated synthesizer (Intavis MultiPep RS)
on modified cellulose disks which are dissolved after synthesis.
The solutions of individual peptides covalently linked to
macromolecular cellulose are then spotted onto coated microscope
slides. The CelluSpots.TM. synthesis was carried out stepwise
utilizing 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on
amino-modified cellulose disks in a. 384-well synthesis plate. In
each coupling cycle, the corresponding amino acids were activated
with a solution of DIGHOBt in DMF. Between coupling steps
un-reacted amino groups were capped with a mixture of acetic
anhydride, diisopropylethyl amine and 1-hydroxybenzotriazole. Upon
completion of the synthesis, the cellulose disks were transferred
to a 96-well plate and treated with a mixture of trifluoroacetic
acid (TEA), dichloromethane, triisoproylsilane (TIS) and water for
side chain deprotection. After removal of the cleavage solution,
the cellulose bound peptides are dissolved with a mixture of TFA,
TFMSA, TIS and water, precipitated with diisopropyl ether and
re-suspended in DMSO. The peptide solutions were subsequently
spotted onto Intavis CelluSpots.TM. slides using an Intavis slide
spotting robot.
For epitope analysis, the slides prepared as described above were
washed with ethanol and then with Tris-buffered saline (TBS; 50 mM
Tris, 137 mM NaCl, 2.7 mM KCl, pH 8) before blocking for 16 h at
4.degree. C. with 5 mL 10.times. Western Blocking Reagent (Roche
Applied Science), 2.5 g sucrose in TBS, 0.1% Tween 20. The slide
was washed with TBS and 0.1% Tween 20 and incubated afterward with
1 .mu.g/mL of the corresponding IGF1 antibodies in TBS and 0.1%
Tween 20 at ambient temperature for 2 h and subsequently washed
with TBS+0.1% Tween 20. For detection, the slide was incubated with
anti-rabbit/anti-mouse secondary HRP-antibody (1:20000 in TBS-T)
followed by incubation with chemiluminescence substrate luminol and
visualized with a Lumilmager (Roche Applied Science).
ELISA-positive SPOTs were quantified and through assignment of the
corresponding peptide sequences the antibody binding epitopes were
identified.
As depicted in FIG. 12, M-05-74 shows a HER3 ECD epitope with the
amino acid sequence VYNKLTFQLEP (SEQ ID NO:43) and a
crossreactivity to a HER4 ECD epitope with the amino acid sequence
VYNPTTFQLE (SEQ ID NO:44) with no detectable signals versus the
hairpin motives in EGFR and the HER2 ECD. No signals at all were
detectable with the 8B8 antibody, therefore the 8B8 antibody
targets epitopes, different from the hairpin peptide motives.
M-08-11 shows a HER3 ECD specific epitope with the amino acid
sequence PLVYNKLTFQLE (corresponding to amino acid residues 3-14 of
SEQ ID NO:1) with no crossreactivity detectable to the other
hairpin sequences of the Her-family.
In FIG. 13, the amino acids identified by Ala-Scan which are
contributing most to the binding of antiHER3/HER4 antibody M-05-74
to its HER3 ECD binding epitope VYNKLTFQLEP (SEQ ID NO:43) and to
its HER4 ECD binding epitope VYNPTTFQLE (SEQ ID NO:44) are
underlined/bold.
Example 5
Binding of HRG to HER3-ECD in the Presence of HER3 Antibody
(ELISA)
A Streptavidin-coated 96-well plate was incubated at 4.degree. C.
with cell culture supernatant containing SBP-tagged HER3-ECD. On
the next day the wells were washed three times with washing buffer
(PBS+0.05% Tween-20) and blocked with PBS containing 1% BSA for one
hour. After another three washes with washing buffer, 40 .mu.l
antibody solution (in Delfia Binding Buffer) was added to each well
as a 2.times. stock of the desired final concentrations (10.sup.-3
to 10.sup.3 nM, alternatively 10.sup.-4 to 10.sup.2 nM).
Immediately 40 .mu.l of 20 nM Europium-labeled Heregulin-beta
(PeproTech, Cat. #100-03) was added to achieve a final
concentration of 10 nM. The plates were incubated on a shaker at
room temperature for two hours. Following three washes with
Delfia.RTM. Wash Buffer, Delfia.RTM. Enhancement Solution was added
and incubated on a shaker for 15 minutes (light protected).
Finally, the plates were measured in a Tecan Infinite F200 reader
using a time-resolved fluorescence measurement protocol. The
binding of M-05-74 (named M-074 in FIG. 14) can promote binding of
HRG to HER3-ECD until a plateau is reached at a signal of 650.
Results are shown in FIG. 14.
Example 6
a) Inhibition of HER3 Phosphorylation in ZR-75-1 Cells
Assays were performed in ZR-75-1 cells according to the following
protocol: Seed cells with 500,000 cells/well into Poly-D-Lysine
coated 6-well plate in RPMI1640 medium with 10% FCS. Incubate for
24 h. Remove medium by aspirating, incubate overnight with 500
.mu.l/well RPMI 1640 with 0.5% FCS. Add antibodies in 500 .mu.l
RPMI 1640 with 0.5% FCS. Incubate for 1 h. Add Heregulin-beta
(PeproTech, Cat. #100-03)) (final concentration 500 ng/ml) for 10
min. To lyse the cells remove medium and add 80 .mu.l ice cold
Triton-X-100 cell lysis buffer and incubate for 5 minutes on ice.
After transferring the lysate into 1.5 ml reaction tube and
centrifugation at 14000 rpm for 15 min at 4.degree. C., transfer
supernatant into fresh reaction tubes. Samples containing equal
amounts of protein in SDS loading buffer were separated on SDS PAGE
and blotted by using a semi-dry Western Blot to nitrocellulose
membranes. Membranes were blocked by 1.times.NET-buffer+0.25%
gelatine for 1 h hour and pHER3 is detected by the antibody
.alpha.Phospho-HER3/ErbB3 (Tyr1289) (21D3), Cell Signaling, #4791
and HER3 by the antibody .alpha.ErbB3 (C-17), Santa Cruz, # sc-285
respectively. After washing and detection of the signals by an POD
coupled secondary antibody, bands were densometricaly scanned.
Percent (%) inhibition of anti-HER3 antibodies M-05-74 on receptor
phosphorylation in zr-75-1 cells is shown below in Table 7.
TABLE-US-00020 TABLE 7 % Inhibition of HER3 phosphorylation in
ZR-75-1 cells pHER3 % inhibition antibody [10 .mu.g/ml] Ctrl 0
M-05-74 49
b) Inhibition of HER3 Phosphorylation of the Bivalent Parent
M-05-74 and the Fab Fragment of M-05-74 (Fab-74)
MCF-7 cells were seeded into 24-Well-plates (1 ml RPMI, 10% FCS,
3.times.105 cells per well) and were incubated at 37.degree. C./5%
CO2 overnight. After 24 hours the media was replaced with 1 ml
media containing 0.5% FCS. After 48 hours the antibodies were added
to a final concentration of 10 .mu.g/ml, 1 .mu.g/ml and 0.1
.mu.g/ml (M-05-74) and 6.66 .mu.g/ml, 0.66 .mu.g/ml and 0.066
.mu.g/ml (Fab-074). The plates were incubated at 37.degree. C. for
50 minutes and then Heregulin-beta (PeproTech, Cat. #100-03) was
added to a final concentration of 500 ng/ml. The plates were
incubated for a further 10 minutes at 37.degree. C./5% CO2. The
cells were washed with PBS and lysed in 40 .mu.l Triton Lysis
Buffer (1% Triton) containing Aprotinin (10 .mu.g/ml),
Orthovanadate (0.4 mM), Phenylmethylsulfonyl fluoride (1 mM). 26
.mu.l of the collected lysates were transferred to reaction tubes
and 14 .mu.l Sample Buffer (NuPAGE LDS Sample Buffer 4.times.,
NuPAGE Sample Reducing Agent 10.times.) was added. The samples were
incubated for 10 minutes at 70.degree. C. and then analysed by
SDS-PAGE (NuPAGE, 4-12% Bis-Tris-Mini-Gel). Electroblotting was
performed using the iBlot Dry Blotting System (Invitrogen). The
nitrocellulose membrane was incubated with phosphoHER3 antibody (a
Phospho Her3, Cellsignaling #4791, Rabbit 1:1000) followed by
incubation with HRP-conjugated secondary antibody (goat anti rabbit
1:5000, BioRad cat: 170-6515). Signal was developed using ECL
Detection Reagents (Amersham RPN2209) on X-Ray film (Roche
Lumi-Film Chemiluminescent Detection Film 11666657001). The
anti-HER3 antibody M-05-74 (full length purified from hybridoma)
and the Fab fragment of the antibody Fab-74 (obtained py papain
cleavage from full length M-05-74) were investigated in eqimolar
amounts. Fab fragments were generated by papain digestion of the
antibody. Briefly, 1 ml of app. 2 mg/ml antibody containing
solution was supplemented with 25 mM Cystein and 70 .mu.g papain
(Roche). After incubation at 37.degree. C. for 1.5 h, the digestion
reaction was stopped by addition of iodoacetamide and the reaction
mixture was purified by MabSelect SuRe.TM. (GE Healthcare). The Fab
containing flowthrough fraction was further purified by size
exclusion chromatography (Superdex.TM. 200; GE Healthcare).
Percent (%) inhibition of anti-HER3 antibodies on receptor
phosphorylation in MCF7 cells is summarised below and in Table 8.
The antibody M-05-74 (full length from hybridoma) and the Fab
fragment of this antibody Fab-74 can inhibit HER3 phosphorylation
in equimolar concentrations to an comparable extent.
TABLE-US-00021 TABLE 8 % Inhibition of HER3 phosphorylation in
MCF-7 cells pHER3 pHER3 % inhibition % inhibition Antibody [6.66
nM] [0.66 nM] control 0 0 M-05-74 94 13 (full length from
hybridoma) Fab 96 14 fragment of M-05-74 (Fab-74)
Example 7
Inhibition of HER2/HER3 Heterodimers (Imunoprecipitation and
Western Blot) in MCF7 Cells
MCF-7 cells were seeded into 6-Well-plates (2 ml RPMI, 10% FCS,
8.times.105 cells per well) and were grown overnight. On the next
day the media was exchanged by 2 ml starving media containing 0.5%
FCS. On day three the antibodies were added to a final
concentration of 10 .mu.g/ml and the plates were incubated at
37.degree. C. After 50 minutes Heregulin-beta (PeproTech, Cat.
#100-03) was added to a final concentration of 500 ng/ml and the
plates were incubated for another 10 minutes at 37.degree. C. The
cells were washed with PBS and lysed in 250 .mu.l Triton Lysis
Buffer containing 1% Digitonin. 60 .mu.l of the collected lysates
were transferred to reaction tubes and incubated with 40 .mu.l
antibody-coupled Sepharose.RTM. (either Herceptin.RTM. or
HER3-antibody #208) and 500 .mu.l Buffer containing 0.3% Digitonin.
The reaction mixes were incubated on a wheel rotator overnight at
4.degree. C. On the next day the reaction mixes were washed three
times with 500 .mu.l Buffer containing 0.3% Digitonin. After the
last wash the supernatant was discarded and 10 .mu.l 4.times.
Loading Buffer was added. The tubes were incubated for 10 minutes
at 70.degree. C. and the supernatants were consequently loaded onto
a gel for SDS-PAGE. After the following Semi-Dry Western Blot the
membranes containing the samples immunoprecipitated with HER2
antibody were incubated with anti-HER3/HER4 antibody M-05-74 (M-074
in FIG. 15), and vice versa. The membranes were then incubated with
HRP-conjugated secondary antibody and the ECL signal was
transferred onto X-Ray film. Results are shown in FIG. 15, showing
a strong inhibition of the HER2/HER heterodimer formation (HER2/HER
heterodimerization) by the M-05-74.
Example 8
Inhibition of Tumor Cell Proliferation of M-05-74 in MDA-MB-175
Cells.
The anti-tumor efficacy of HER3 antibodies M-05-74 in a cell
proliferation assay, using MDA-MB-175 cells (VII Human Breast
Carcinoma Cells, ATCC catalog no. HTB-25), was assessed. 20,000
cells per well were seeded into sterile 96 well tissue culture
plates with DMEM/F12 cell culture medium, containing 10% FCS and
incubated at 37.degree. C..+-.1.degree. C. with 5%.+-.1% CO.sub.2
for one day. The cells are slow growing cells with a doubling time
of ca. 3 days. Anti-HER3 antibodies were added in dilution series
and further incubated for 6 days. Cell viability was then assessed
using the alamarBlue.RTM. readout. EC50 values were calculated.
TABLE-US-00022 TABLE 9 EC50 of the Inhibition of tumor cell
proliferation of M-05-74 in MDA- MB-175 cells antibody EC.sub.50
[.mu.g/ml] M-05-74 5.8
Example 9
In Vivo Antitumor Efficacy of Anti-HER3 Antibody M-05-74
The in vivo antitumor efficacy of the anti-HER3 antibody M-05-74
(M-074) could be detected in cell based models of various tumor
origin (e.g. SCCHN and pancreatic cancer) transplanted on SCID
beige. As example data are shown for the SCCHN xenograft model FaDu
(cell line based).
Test Agents
M-05-74 was provided as stock solution from Roche, Penzberg,
Germany expressed and purified from hybridoma cells. Antibody
buffer included histidine. Antibody solution was diluted
appropriately in buffer from stock prior injections.
Cell Lines and Culture Conditions
FaDu human HNSCC cells were originally obtained from ATCC. The
tumor cell line was routinely cultured in MEM Eagle medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM
sodium pyruvate and 0.1 mM NEAA at 37.degree. C. in a
water-saturated atmosphere at 5% CO2. Culture passage was performed
with trypsin/EDTA 1.times. splitting every third day.
Animals
Female SCID beige or nude mice were purchased from breeder (e.g.
Charles River, Sulzfeld, Germany) and maintained under
specific-pathogen-free condition with daily cycles of 12 h light/12
h darkness according to committed guidelines (GV-Solas; Felasa;
TierschG). Experimental study protocol was reviewed and approved by
local government. After arrival animals were maintained in the
quarantine part of the animal facility for one week to get
accustomed to new environment and for observation. Continuous
health monitoring was carried out on regular basis. Diet food
(Provimi Kliba 3337) and water (acidified pH 2.5-3) were provided
ad libitum.
Animals were controlled daily for clinical symptoms and detection
of adverse effects. For monitoring throughout the experiment body
weight of animals was documented.
Animal treatment started after animal randomisation after cell
transplantation when median tumor size was about 100-150 mm3.
Antibody was administered as single agent at 10 mg/kg i.p. q7d once
weekly for several weeks depending of the model. The corresponding
vehicle was administered on the same days.
FaDu HNSCC xenograft bearing mice were treated with antibody
M-05-74 from study day 10 to 24. As a result, treatment with H-74
antibody showed significant anti-tumor efficacy with nearly tumors
stasis of s.c. FaDu xenografts. The Tumor Growth Inhibition (TGI)
was calculated at 89%.
Treatment with M-05-74 (10 mg/kg q7d.times.3, i.p.) resulted in
nearly tumor stasis of FaDu. Results are shown in FIG. 17, wherein
M-05-74 is named M-074.
Example 10
Generation of M-05-74-Fab-Pseudomonas Exotoxin Conjugate
(M-05-74-PE)
Expression, purification and renaturation of Fab fragment of
M-05-74, PE24 variant, and Fab fragment of M-05-74 conjugated to
Pseudomonas exotoxin variant PE24LR8M based on the Sequences of SEQ
ID NO:45, 46, 47, 48 (or 49).
Expression of Fab (e.g. for Sortase Coupling)--Expression
Vectors
For the expression of the described Fab fragments, variants of
expression plasmids for transient expression (e.g. HEK293-F) cells
based either on a cDNA organization with or without a CMV-Intron A
promoter or on a genomic organization with a CMV promoter were
applied.
Beside the antibody expression cassette the vectors contained: an
origin of replication which allows replication of this plasmid in
E. coli, and a -lactamase gene which confers ampicillin resistance
in E. coli.
The transcription unit of the antibody gene was composed of the
following elements: unique restriction site(s) at the 5' end the
immediate early enhancer and promoter from the human
cytomegalovirus, followed by the Intron A sequence in the case of
the cDNA organization, a 5'-untranslated region of a human antibody
gene, an immunoglobulin heavy chain signal sequence, the human
antibody chain either as cDNA or as genomic organization with the
immunoglobulin exon-intron organization a 3' untranslated region
with a polyadenylation signal sequence, and unique restriction
site(s) at the 3' end.
The fusion genes comprising the antibody chains as described below
were generated by PCR and/or gene synthesis and assembled by known
recombinant methods and techniques by connection of the according
nucleic acid segments e.g. using unique restriction sites in the
respective vectors. The subcloned nucleic acid sequences were
verified by DNA sequencing. For transient transfections larger
quantities of the plasmids were prepared by plasmid preparation
from transformed E. coli cultures (Nucleobond AX,
Macherey-Nagel).
Cell Culture Techniques
Standard cell culture techniques were used as described in Current
Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M.,
Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.),
John Wiley & Sons, Inc.
The Fab fragments were expressed by transient co-transfection of
the expression plasmids of the heavy and the light chain in HEK29-F
cells growing in suspension as described below.
Transient Transfections in HEK293-F System
The Fab fragments were generated by transient transfection with the
respective plasmids (e.g. encoding the heavy and modified heavy
chain, as well as the corresponding light and modified light chain)
using the HEK293-F system (Invitrogen) according to the
manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen)
growing in suspension either in a shake flask or in a stirred
fermenter in serum-free FreeStyle.TM. 293 expression medium
(Invitrogen) were transfected with a mix of the four expression
plasmids and 293-Free.TM. (Novagen) or Fectin (Invitrogen). For 2 L
shake flask (Corning) HEK293-F cells were seeded at a density of
1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day
after the cells were transfected at a cell density of ca. 1.5E*6
cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM.TM. (Invitrogen)
with 600 .mu.g total plasmid DNA (1 .mu.g/mL) encoding the heavy or
modified heavy chain, respectively and the corresponding light
chain in an equimolar ratio and B) 20 ml Opti-MEM.TM.+1.2 mL
293-Free.TM. (Novagen) or Fectin (2 .mu.l/mL). According to the
glucose consumption glucose solution was added during the course of
the fermentation. The supernatant containing the secreted antibody
was harvested after 5-10 days and antibodies were either directly
purified from the supernatant or the supernatant was frozen and
stored.
Expression of Pseudomonas Exotoxin Variant PE24-LR8M for Sortase
Coupling--Expression Vector
For the expression of PE24-LR8M an E. coli expression plasmid was
used.
Beside the expression cassette for the pseudomonas exotoxin A
domain III the vector contained: an origin of replication from the
vector pBR322 for replication in E. coli (according to Sutcliffe,
G., et al., Quant. Biol. 43 (1979) 77-90), the lacI repressor gene
from E. coli (Farabaugh, P. J., Nature 274 (1978) 765-769), the
URA3 gene of Saccharomyces cerevisiae coding for orotidine
5'-phosphate decarboxylase (Rose, M. et al. Gene 29 (1984) 113-124)
which allows plasmid selection by complementation of E. coli pyrF
deletion strains (uracil auxotrophy).
The transcription unit of the toxin gene was composed of the
following elements: unique restriction site(s) at the 5' end, the
T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according to
Bujard, H., et al. Methods. Enzymol. 155 (1987) 416-433 and
Stueber, D., et al., Immunol. Methods IV (1990) 121-152) including
a synthetic ribosomal binding site according to Stueber, D., et al.
(see before), the pseudomonas exotoxin A domainIII with an
N-terminal coupling tag followed by a furin site (SEQ ID NO:45
Pseudomonas exotoxin variant PE24LR8M_3G, including a GGG linker
for sortase coupling), two bacteriophage-derived transcription
terminators, the .lamda.-T0 terminator (Schwarz, E., et al., Nature
272 (1978) 410-414) and the fd-terminator (Beck E. and Zink, B.
Gene 1-3 (1981) 35-58), unique restriction site(s) at the 3' end.
Cultivation and Expression of the Pseudomonas Exotoxin A Construct
Variant PE24-LR8M_3G in an E. coli Fed-Batch Process on Chemical
Defined Medium
For the expression of PE24-LR8M_3G_Ecoli (25 kDa) the E. coli
host/vector system which enables an antibiotic-free plasmid
selection by complementation of an E. coli auxotrophy (PyrF) was
employed (EP 0 972 838 and U.S. Pat. No. 6,291,245).
An E. coli K12 strain was transformed by electroporation with the
expression plasmid. The transformed E. coli cells were first grown
at 37.degree. C. on agar plates. A colony picked from this plate
was transferred to a 3 mL roller culture and grown at 37.degree. C.
to an optical density of 1-2 (measured at 578 nm). Then 1000 .mu.l
culture where mixed with 1000 .mu.l sterile 86%-glycerol and
immediately frozen at -80.degree. C. for long time storage. The
correct product expression of this clone was first verified in
small scale shake flask experiments and analyzed with SDS-Page
prior to the transfer to the 10 L fermenter.
Pre Cultivation:
For pre-fermentation a chemical defined medium has been used. For
pre-fermentation 220 ml of medium in a 1000 ml Erlenmeyer-flask
with four baffles was inoculated with 1.0 ml out of a primary seed
bank ampoule. The cultivation was performed on a rotary shaker for
8 hours at 32.degree. C. and 170 rpm until an optical density (578
nm) of 2.9 was obtained. 100 ml of the pre cultivation was used to
inoculate the batch medium of the 10 L bioreactor.
Fermentation:
For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,
Melsungen, Germany) a chemical defined batch medium was used. All
components were dissolved in deionized water. The alkaline solution
for pH regulation was an aqueous 12.5% (w/v) NH.sub.3 solution
supplemented with 11.25 g/l L-methionine.
Starting with 4.2 l sterile batch medium plus 100 ml inoculum from
the pre cultivation the batch fermentation was performed at
31.degree. C., pH 6.9.+-.0.2, 800 mbar back pressure and an initial
aeration rate of 10 l/min. The relative value of dissolved oxygen
(pO2) was kept at 50% throughout the fermentation by increasing the
stirrer speed up to 1500 rpm. After the initially supplemented
glucose was depleted, indicated by a steep increase in dissolved
oxygen values, the temperature was shifted to 25.degree. C. and 15
minutes later the fermentation entered the fed-batch mode with the
start of both feeds (60 and 14 g/h respectively). The rate of feed
2 is kept constant, while the rate of feed 1 is increased stepwise
with a predefined feeding profile from 60 to finally 160 g/h within
7 hours. When carbon dioxide off gas concentration leveled above 2%
the aeration rate was constantly increased from 10 to 20 l/min
within 5 hours. The expression of recombinant PE24-LR8M_3G_Ecoli
protein was induced by the addition of 2.4 g IPTG at an optical
density of approx. 120. The target protein is expressed soluble
within the cytoplasm.
After 24 hours of cultivation an optical density of 209 is achieved
and the whole broth is cooled down to 4-8.degree. C. The bacteria
are harvested via centrifugation with a flow-through centrifuge
(13,000 rpm, 13 l/h) and the obtained biomass is stored at
-20.degree. C. until further processing (cell disruption). The
yield is 67.5 g dry cells per liter.
Analysis of Product Formation:
Samples drawn from the fermenter, one prior to induction and the
others at dedicated time points after induction of protein
expression are analyzed with SDS-Polyacrylamide gel
electrophoresis. From every sample the same amount of cells
(OD.sub.Target=10) are suspended in 5 mL PBS buffer and disrupted
via sonication on ice. Then 100 .mu.L of each suspension are
centrifuged (15,000 rpm, 5 minutes) and each supernatant is
withdrawn and transferred to a separate vial. This is to
discriminate between soluble and insoluble expressed target
protein. To each supernatant (=soluble protein fraction) 100 .mu.L
and to each pellet (=insoluble protein fraction) 200 .mu.L of SDS
sample buffer (Laemmli, U. K., Nature 227 (1970) 680-685) are
added. Samples are heated for 15 minutes at 95.degree. C. under
intense mixing to solubilize and reduce all proteins in the
samples. After cooling to room temperature 5 .mu.L of each sample
are transferred to a 4-20% TGX Criterion Stain Free polyacrylamide
gel (Bio-Rad). Additionally 5 .mu.l molecular weight standard
(Precision Plus Protein Standard, Bio-Rad) were applied.
The electrophoresis was run for 60 Minutes at 200 V and thereafter
the gel was transferred the GelDOC.TM. EZ Imager (Bio-Rad) and
processed for 5 minutes with UV radiation. Gel images were analyzed
using Image Lab analysis software (Bio-Rad). Relative
quantification of protein expression was done by comparing the
volume of the product bands to the volume of the 25 kDa band of the
molecular weight standard.
Cultivation and Expression of an Antibody Fragment Light Chain
Construct (VL) and an Antibody Fragment Heavy Chain Pseudomonas
Exotoxin A Variant Fusion (Fab-PE24) in an E. coli Fed-Batch
Process on Chemical Defined Medium
For the expression of a Fab-light chain (23.4 kDa) and a Fab-heavy
chain PE24 fusion (48.7 kDa) the E. coli host/vector system which
enables an antibiotic-free plasmid selection by complementation of
an E. coli auxotrophy (PyrF) was employed (EP 0 972 838 and U.S.
Pat. No. 6,291,245).
An E. coli K12 strain was transformed by electroporation with the
respective expression plasmids. The transformed E. coli cells were
first grown at 37.degree. C. on agar plates. For each
transformation a colony picked from this plate was transferred to a
3 mL roller culture and grown at 37.degree. C. to an optical
density of 1-2 (measured at 578 nm). Then 1000 .mu.l culture where
mixed with 1000 .mu.l sterile 86%-glycerol and immediately frozen
at -80.degree. C. for long time storage. The correct product
expression of these clones was first verified in small scale shake
flask experiments and analyzed with SDS-Page prior to the transfer
to the 10 L fermenter.
Pre-Cultivation:
For pre-fermentation a chemical defined medium has been used. For
pre-fermentation 220 ml of medium in a 1000 ml Erlenmeyer-flask
with four baffles was inoculated with 1.0 ml out of a primary seed
bank ampoule. The cultivation was performed on a rotary shaker for
9 hours at 37.degree. C. and 170 rpm until an optical density (578
nm) of 7 to 8 was obtained. 100 ml of the pre cultivation was used
to inoculate the batch medium of the 10 L bioreactor.
Fermentation (RC52#003):
For fermentation in a 101 Biostat C, DCU3 fermenter (Sartorius,
Melsungen, Germany) a chemical defined batch medium was used. The
alkaline solution for pH regulation was an aqueous 12.5% (w/v)
NH.sub.3 solution supplemented with 11.25 g/l L-methionine.
Starting with 4.2 l sterile batch medium plus 100 ml inoculum from
the pre cultivation the batch fermentation was performed at
31.degree. C., pH 6.9.+-.0.2, 800 mbar back pressure and an initial
aeration rate of 10 l/min. The relative value of dissolved oxygen
(pO2) was kept at 50% throughout the fermentation by increasing the
stirrer speed up to 1500 rpm. After the initially supplemented
glucose was depleted, indicated by a steep increase in dissolved
oxygen values, the temperature was shifted to 37.degree. C. and 15
minutes later the fermentation entered the fed-batch mode with the
start of both feeds (60 and 14 g/h respectively). The rate of feed
2 is kept constant, while the rate of feed 1 is increased stepwise
with a predefined feeding profile from 60 to finally 160 g/h within
7 hours. When carbon dioxide off gas concentration leveled above 2%
the aeration rate was constantly increased from 10 to 20 l/min
within 5 hours. The expression of recombinant target proteins as
insoluble inclusion bodies located in the cytoplasm was induced by
the addition of 2.4 g IPTG at an optical density of approx. 40.
After 24 hours of cultivation an optical density of 185 is achieved
and the whole broth is cooled down to 4-8.degree. C. The bacteria
are harvested via centrifugation with a flow-through centrifuge
(13,000 rpm, 13 l/h) and the obtained biomass is stored at
-20.degree. C. until further processing (cell disruption). The
yield is between 40 and 60 g dry cells per liter.
Analysis of Product Formation:
Samples drawn from the fermenter, one prior to induction and the
others at dedicated time points after induction of protein
expression are analyzed with SDS-Polyacrylamide gel
electrophoresis. From every sample the same amount of cells
(OD.sub.Target=10) are suspended in 5 mL PBS buffer and disrupted
via sonication on ice. Then 100 .mu.L of each suspension are
centrifuged (15,000 rpm, 5 minutes) and each supernatant is
withdrawn and transferred to a separate vial. This is to
discriminate between soluble and insoluble expressed target
protein. To each supernatant (=soluble protein fraction) 100 .mu.L
and to each pellet (=insoluble protein fraction) 200 .mu.L of SDS
sample buffer (Laemmli, U. K., Nature 227 (1970) 680-685) are
added. Samples are heated for 15 minutes at 95.degree. C. under
intense mixing to solubilize and reduce all proteins in the
samples. After cooling to room temperature 5 .mu.L of each sample
are transferred to a 4-20% TGX Criterion Stain Free polyacrylamide
gel (Bio-Rad). Additionally 5 .mu.l molecular weight standard
(Precision Plus Protein Standard, Bio-Rad) and 3 amounts (0.3
.mu.l, 0.6 .mu.l and 0.9 .mu.l) quantification standard with known
target protein concentration (0.1 .mu.g/.mu.l) were applied.
The electrophoresis was run for 60 Minutes at 200 V and thereafter
the gel was transferred the GelDOC.TM. EZ Imager (Bio-Rad) and
processed for 5 minutes with UV radiation. Gel images were analyzed
using Image Lab analysis software (Bio-Rad). With the three
standards a linear regression curve was calculated with a
coefficient of >0.99 and thereof the concentrations of target
protein in the original sample was calculated.
Purification, Sortase Coupling and Renaturation (of Fab Fragment of
M-05-74, PE24 Variant, and Fab Fragment of M-05-74 Conjugated to
Pseudomonas Exotoxin Variant PE24LR8M)
Fab Fragment
The Fab fragment was purified by affinity chromatography (Ni
Sepharose.RTM. High Perfomance HisTrap.TM.) according to the
manufacture's description. In brief, the supernatant was loaded
onto the column equilibrated in 50 mM sodium phosphate pH 8.0, 300
mM NaCl. Protein elution was performed with the same buffer at pH
7.0 with a washing step containing 4 mM imidazole followed by a
gradient up to 100 mM imidazole. Fractions containing the desired
Fab fragment were pooled and dialyzed against 20 mM His, 140 mM
NaCl, pH 6.0.
PE24 for Sortase Coupling
E. coli cells expressing PE24 were lysed by high pressure
homogenization (if details are required: Christian Schantz) in 20
mM Tris, 2 mM EDTA, pH 8.0+Complete protease inhibitor cocktail
tablets (Roche). The lysate was filtrated and loaded onto a Q
Sepharose.RTM. FF (GE Healthcare) equilibrated in 20 mM Tris, pH
7.4. Protein was eluted with a gradient up to 500 mM NaCl in the
same buffer. PE24 containing fractions were identified by SDS PAGE.
The combined pool was concentrated and applied to a HiLoad.TM.
Superdex.TM. 75 (GE Healthcare) equilibrated in 20 mM Tris, 150 mM
NaCl, pH 7.4. Fractions containing PE24 were pooled according to
SDS PAGE and frozen at -80.degree. C.
Sortase Coupling of Fab Fragment to PE24
Fab fragment and PE24 were diafiltrated separately into 50 mM Tris,
150 mM NaCl, 5 mM CaCl.sub.2) pH7.5 using Amicon.RTM. Ultra 4
centrifugal filter devices (Merck Millipore) and concentrated to
5-10 mg/ml. Both proteins and sortase were combined in a 1:1:0.8
molar ratio. After one hour incubation at 37.degree. C. the mixture
was loaded onto a Ni Sepharose.RTM. High Perfomance HisTrap.TM.)
equilibrated in 50 mM sodium phosphate, pH 8.0, 300 mM NaCl.
Elution was performed with a gradient up to 100 mM imidazole in the
same buffer pH 7.0. The flow through fractions containing the final
product Fab-PE24 was concentrated and loaded onto a HiLoad.TM.
Superdex.TM. 200 (GE Healthcare) in 20 mM Tris, 150 mM NaCl, pH
7.4. Fractions containing the desired coupled protein were pooled
and stored at -80.degree. C. As sortase soluble S. aureus sortase A
was used (SEQ ID NO: 50). Soluble S. aureus sortase A was expressed
and purified using the following expression plasmid: The sortase
gene encodes an N-terminally truncated Staphylococcus aureus
sortase A (60-206) molecule. The expression plasmid for the
transient expression of soluble sortase in HEK293 cells comprised
besides the soluble sortase expression cassette an origin of
replication from the vector pUC18, which allows replication of this
plasmid in E. coli, and a beta-lactamase gene which confers
ampicillin resistance in E. coli. The transcription unit of the
soluble sortase comprises the following functional elements: the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, a human heavy chain
immunoglobulin 5'-untranslated region (5'UTR), a murine
immunoglobulin heavy chain signal sequence, an N-terminally
truncated S. aureus sortase A encoding nucleic acid, and the bovine
growth hormone polyadenylation sequence (BGH pA). Renaturation of
Fab-PE24 Derived from E. coli Inclusion Bodies
Inclusion bodies of VH-PE24 and VL-C.sub.kappa were solubilized
separately in 8 M guanidinium hydrochloride, 100 mM Tris-HCl, 1 mM
EDTA, pH 8.0+100 mM dithiothreitol (DTT). After 12-16 hours at RT
the pH of the solubilisates was adjusted to 3.0, the centrifuged
solutions were dialyzed against 8 M guanidinium hydrochloride, 10
mM EDTA, pH 3.0. The protein concentration was determined by Biuret
reaction, the purity of inclusion body preparations was estimated
by SDS PAGE. Equimolar amounts of both chains were diluted in two
steps into 0.5 M arginine, 2 mM EDTA, pH 10+1 mM GSH/1 mM GSSG, to
a final concentration of 0.2-0.3 mg/ml. After 12-16 h at
4-10.degree. C. the renaturated protein was diluted with H.sub.2O
to <3 mS/cm and loaded onto a Q Sepharose.RTM. FF (GE
healthcare) equilibrated in 20 mM Tris/HCl, pH 7.4. Elution was
performed with a gradient up to 400 mM NaCl in the same buffer.
Fractions containing the correct product were identified by
SDS-PAGE and analytical size exclusion chromatography (SEC). Pooled
fractions were concentrated and loaded onto a HiLoad.TM.
Superdex.TM. 200 (GE Healthcare) in 20 mM Tris, 150 mM NaCl, pH 7.4
or alternatively in 20 mM histidine, 140 mM NaCl, pH 6.0. Fractions
were analyzed and pooled according to analytical SEC and stored at
-80.degree. C.
Based on SEQ ID NO:46 and 49 the immunoconjugate of Fab fragment of
M-05-74 with Pseudomonas exotoxin variant PE24LR8M (M-05-74-PE) can
be expressed recombinately, purified and renaturated also as direct
PE24LR8M fusion.
Example 11
Cell Killing of Different Tumor Cell Lines by
M-05-74-Fab-Pseudomonas Exotoxin Conjugate (M-05-74-PE)
HER3 overexpressing A549 cells were seeded into a white
96-well-plate (flat, transparent bottom, 1.times.10.sup.4 cells per
well) and were grown in RPMI (10% FCS) overnight. On the next day,
the media was exchanged by 50 .mu.l starving media (RPMI, 0.5%
FCS). After at least 4 hours, 5 .mu.l Heregulin-beta (PeproTech,
Cat. #100-03) (HRG beta) was added to a final concentration of 500
ng/ml. 50 .mu.l Fab-74-PE solution was added to final
concentrations of 10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.005 and
0.002 .mu.g/ml. Plates were incubated for 72 h. After 24 h and 48
h, 5 .mu.l Heregulin-beta was added again to a final concentration
of 500 ng/ml. After 72 h the luminescence was measured in a Tecan
Infinite.RTM. F200 Reader using the CellTiter-Glo.RTM. Luminescent
Cell Viability Assay by Promega (Cat. # G7571). The EC50 value for
M-05-74-Fab-Pseudomonas exotoxin conjugate (M-05-74-PE) in the
absence of HRG beta was: 1.93 .mu.g/ml and in the presence 0.13
.mu.g/ml.
TABLE-US-00023 TABLE 10 EC50 of Cell killing of A549 cells by
M-05-74-Fab-Pseudomonas presence (+)/ absence (-) of ligand EC50 of
(M- Heregulin-beta 05-74-PE) (HRG) (.mu.g/ml) half max. inhibition
+HRG beta 0.13 30.3 -HRG beta 1.93 19.85
Example 12a
Humanized Variants of Anti-HER3 Antibody M-05-74
The murine antibody M-05-7 heavy chain and light chain variable
domains were used to search for similar human antibody variable
domains. From the 200 results obtained for each chain about half
were rejected as being from a non-human source. All of the
remaining human antibodies were analyzed for key residues within
the frameworks that are involved in the VH/VL interface, and for
residues that are important for the CDR loop structure. As far as
possible these key residues important for the VH/VL interface and
canonical loop structure have been maintained in the humanized
variants, however certain changes of these positions are included
sometimes. The CDRs from the murine antibody chains were grafted
into these human antibody frameworks. The top five grafted domains
were chosen based upon the previous criteria and also on the
results of a T-cell epitope in silico screen for further
development. Accordingly the mouse anti-HER3 antibody M-05-74 was
humanized to give the following humanized variant VH and VL domains
of M-05-74:
TABLE-US-00024 TABLE 11 VH and VL sequences of humanized variant
antibodies of M-05-74 humanized variant of light humanized variant
of VH/SEQ ID chain variable domain VL//SEQ NO: ID NO: <Her3>
M-05-74_VH-A <Her3> M-05-74_VL-A SEQ ID NO: 33 SEQ ID NO: 38
<Her3> M-05-74_VH-B <Her3> M-05-74_VL-B SEQ ID NO: 34
SEQ ID NO: 39 <Her3> M-05-74_VH-C <Her3> M-05-74_VL-C
SEQ ID NO: 35 SEQ ID NO: 40 <Her3> M-05-74_VH-D <Her3>
M-05-74_VL_D SEQ ID NO: 36 SEQ ID NO: 41 <Her3> M-05-74_VH-E
<Her3> M-05-74_VL-E SEQ ID NO: 37 SEQ ID NO: 42
From the 25 theoretically possible combinations of these five VH
and VL domains the most potent binders were selected as
follows:
In order to find a most optimized humanized variant of the
<Her3> M-05-74 antibody with the favorable kinetic
properties, five variants of each heavy and light chain were
designed as described above. The obtained sequences were generated
in all combinations (25 in total) in a scFv-ribosome display
construct.
The 25 scFv constructs were amplified by flanking primers to obtain
linear template DNA, necessary for ribosome display. Each PCR
product was purified with agarose gel-electrophoresis followed by
extraction with the Qiagen MinElute.TM. Kit according to the
manufacturer's instructions. The product DNA concentration was
determined and 200 ng of an equimolar mixture of all linear
template DNAs was the basis for the in-vitro
transcription/translation at 37.degree. C. for 60 min. The utilized
kit comprised the PURExpress.RTM. in-vitro protein synthesis kit
(NEB), including both disulfide bond enhancers (DBE 1 & 2). Two
reaction samples were processed, with the doubled reaction amount
per sample. The first sample included the biotinylated and
heregulin activated target (Her3-ECD) in the subsequent panning
step. The second sample was the negative control, without target
protein in the panning step. Both samples were treated identically.
The obtained pools of ternary complexes (mRNA-ribosome-scFv
variant) after transcription and translation were subjected to a
pre-panning step with the employed magnetic beads (Streptavidin
M-270 Dynabeads, Life Technologies) for 30 min at 4.degree. C. to
remove unspecific binding variants. The pre-panning beads were
removed by centrifugation and the supernatant with the remaining
ternary complexes was added to the prepared target/heregulin
mixture to incubate for 30 min at 4.degree. C. in the panning step.
The target/heregulin complex was incubated in a 1:6 molar ratio for
60 min previous to the panning step to obtain the open conformation
of the receptor domain and to expose the epitope of the 74 parental
antibody. The final concentration of biotinylated Her3-ECD in the
panning reaction was 100 nM. All employed buffers hereafter
contained 300 nM heregulin.
The target and all binding ternary complexes were captured via the
targets biotin taq and the above mentioned streptavidin beads.
Incubation time for capturing was 20 min at 4.degree. C. Utilizing
the magnetic properties of the beads the complexes can be washed by
repeated incubation and removal of the wash buffer. In order to
remove weak binding variants the wash pressure was increased over
the washing steps. In total five washing steps with 500 uL of wash
buffer (containing Heregulin) were employed (2, 4, 5, 5 & 1
min) with 2 min of capturing in the magnetic field in between. The
last step was used to transfer the remaining strong binding
variants in a clean new reaction tube for the elution step (10 min,
4.degree. C., 100 uL elution buffer containing EDTA) followed by
centrifugation to remove the beads. The obtained RNA in the
supernatant was purified with the Qiagen RNEasy RNA purification
kit according to the manufacturer's instructions. In order to
ensure the origin of the later produced DNA by reverse
transcription, the RNA was beforehand subjected to an DNAse
digestion. The digest (Ambion DNA-free Kit) was initiated with 12
uL of purified RNA and incubated for 30 min at 37.degree. C.
Following the removal of DNase, three reverse transcription
reactions per sample were initiated with 12 uL each and incubated
for one hour at 37.degree. C. 12 uL of each digested RNA sample
(digested product) were used as negative control for the first PCR
to proove the complete removal of DNA traces.
The products of the reverse transcription reactions were pooled for
each sample and used to initiate five 100 uL PCR reactions to
amplify the DNA selection pools. The products were pooled and
purified by gel electrophoresis (1% preparative agarose gel and
analytical Agilent DNA 7500 chip with 1 uL sample volume) and the
Qiagen MinElute Kit according to the manufacturer's protocols. The
obtained gel image in FIG. 1 clearly shows enrichment of selected
construct DNA in lane 1 and no enrichment for the negative
control--panning without target--in lane 2. The remaining controls
are also negative as expected. The DNA digest was complete (lane 3
for target, lane 4 for background). Therefore all obtained DNA in
lane 1 is derived from binding variants, selected in the panning
step, and their corresponding RNA. Neither the negative control of
the reverse transcription, nor the negative control of the PCR is
showing bands. Lane 7 shows the product of the pooled PCR reactions
after purification.
The PCR product was amplified to produce enough DNA for cloning.
The selection pool and the expression vector Her_scFv_huFc (1 ug
each) were digested with MfeI-HF and NotI-HF in CutSmart.RTM.
buffer (all NEB) for one hour at 37.degree. C. The selection insert
and the cut vector were first purified and then ligated with NEB
Quick Ligase for 30 min at room temperature. The molar ratio of cut
insert to vector was 5:1 (25 ng cut insert and 50 ng cut vector).
Two microliters of the ligation product were directly used to
transform 50 uL of DHS.alpha. (Life Technologies) competent cells.
Following outgrowth, 50 uL were plated out on LB plates with
ampicillin resistance (LBamp) and incubated for 16 hours at
37.degree. C. 34 colonies were used to inoculate 5 mL LBamp media
for 16 h at 37.degree. C. The cells were harvested and the DNA
isolated with the Qiagen Miniprep Kit according to the
manufacturer's instructions and 300 ng plasmid DNA of each sample
was sent to Sequiserve GmbH for sequencing.
Results--Most Optimized Humanized Variant of <Her3> M-05-74
Antibody
The sequencing results show an enrichment of one particular
variant: VH-A/VL-D. The corresponding sequence was obtained six
times from the 34 samples, which clearly indicates the most potent
binding properties to HER-ECD in the assay described above.
Also the combinations VH-A/VH-B and VH-A/VH-E occurred twice and
hence showed some superior binding properties to HER3 ECD as
compared to the remaining less enriched VH/VLcombinations.
Surpisingly all enriched variants included VH-A. Consequently VH-A
is a key feature of all HER3 binding humanized variants of
<Her3> M-05-74., especially in the preferred combinations
VH-A/VL-D, VH-A/VH-B and VH-A/VH-E.
The remaining 24 sequences were all different and featured minor
deletions and/or a combination of point mutations. Three sequences
could not perfectly be edited and were not analyzed.
Each of the combinations VH-A/VL-D, VH-A/VH-B and VH-A/VH-E. is
expressed in a human IgG1 isotype (with Ckappa light chain constant
domain) or alternatively e.g. as fusion protein with a Pseudomonas
exotoxin (immunotoxin) as described above. Binding characteristics
and biological properties are determined as describe above e.g. in
Example 2, 3, 5, 6, 7, 8, 9, 11 or described in Example 13
below.
Example 12b
Binding of Humanized Variants of Anti-HER3 Antibody M-05-74
To investigate the binding of the humanized variant VH-A/VL-D of
anti-HER3 antibody M-05-74 (described in Example 12a) to the
HER3-ECD and the HER4-ECD, in presence and absence of the ligand
Heregulin, SPR analysis were conducted at 37.degree. C., using a
Biacore.TM. 3000 device (GE Healthcare) (Table 11).
TABLE-US-00025 TABLE 11a SPR analysis of humanized variant
VH-A/VL-D at 37.degree.C.: Binding of humanized DIB-74 to HER3-ECD
and HER4-ECD in presence and absence of the ligand FRG,
investigated using a Biacore .TM. 3000 device (GE Healthcare)
t.sub./2diss K.sub.D R.sub.max Chi.sup.2 Analyte k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) (min) (nM) (RU) MR (RU.sup.2)
HER3-ECD 1.9E+04 4.4E-04 26 23 80 0.7 0.2 HER4-ECD n. d. n. d. n.
d. n. d. n. d. n. d. n. d. HER3- 1.9E+05 2.0E-03 6 10 198 0.8 1.4
ECD/HRG HER4- 1.8E+05 3.8E-02 0.3 211 183 0.8 0.5 ECD/HRG
TABLE-US-00026 TABLE 11b Direct comparison with parent murine
anti-HER3 antibody M-05-74 t.sub./2diss K.sub.D R.sub.max Chi.sup.2
Analyte k.sub.a (M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) (min) (nM)
(RU) MR (RU.sup.2) HER3-ECD 2.1E+04 8.1E-05 144 4 104 0.6 0.2
HER4-ECD n.d. n.d. n.d. n.d. n.d. n.d. n.d. HER3- 7.9E+04 5.7E-04
20 7 225 0.7 2.6 ECD/HRG HER4- 5.8E+05 3.3E-03 4 6 289 0.9 5.4
ECD/HRG
The humanized variant VH-A/VL-D of anti-HER3 antibody M-05-74
preferentially bound to the ligand activated ECD complexes, due to
increased epitope accessibility. It bound with an affinity of
K.sub.D 10 nM to the HER3-ECD/HRG complex
Surpisingly the humanized variant VH-A/VL-D of anti-HER3 antibody
M-05-74 showed a strongly reduced HER4-ECD/HRG reactivity (K.sub.D
211 nM) compared to the parent antibody M-05-74 ((K.sub.D 4 nM))
while retaining its HER3-ECD/HRG reactivity (K.sub.D 10 nM compared
to K.sub.D 7 nM).
Example 13
In Vivo Tumor Cell Growth Inhibition by M-05-74-Fab-Pseudomonas
Exotoxin Conjugate (M-05-74-PE)
The human A431-B34 non-small cell lung cancer cell line cell line,
which was stably transfected with an expression vector encoding
human HER3, was subcutaneously inoculated into the right flank of
female SCID beige mice (1.times.10.sup.7 cells per animal).
On day 21 after tumor inoculation, the animals were randomized and
allocated into the treatment group and one vehicle group, resulting
in a median tumor volume of .about.110 mm.sup.3 per group. On the
same day, animals were treated intravenously for 2 cycles, each
cycle consisting of 3q7d (every other day), with
M-05-74-Fab-Pseudomonas exotoxin conjugate (M-05-74-PE) (1.0
mg/kg). Controls received vehicle (Tris buffer). The two cycles
were separated by a one week off-treatment.
Primary tumor volume (TV) was calculated according to the NCI
protocol (TV=(length.times.width.sup.2)/2), where "length" and
"width" are long and short diameters of tumor mass in mm (Corbett
et al., 1997). Calculation was executed from staging (day 21 after
tumor inoculation) until day 42 after tumor inoculation, and values
were documented as medians and inter-quartile ranges (IQR) defined
as differences of the third and first quartile.
For calculation of percentage tumor growth inhibition (TGI) during
the treatment period, every treated group was compared with its
respective vehicle control. TV.sub.day z represents the tumor
volume of an individual animal at a defined study day (day z) and
TV.sub.day x represents the tumor volume of an individual animal at
the staging day (day x).
The following formula was applied:
.times..times..times..times..times..times..times..times..times..times..ti-
mes..times..times..times..times..times..times..times..times..times..times.-
.times..times..times..times. ##EQU00001##
Calculations of treatment to control ratio (TCR) with confidence
interval (CI) were applied using non-parametric methods. Results of
median tumor volumes with inter-quartile ranges are shown in FIG.
19. Tumor growth inhibition was 66% of M-05-74-Fab-Pseudomonas
exotoxin conjugate (M-05-74-PE) with a TCR of 0.509
(CI:0.33-0.734).
Example 14
Binding of the Antibody M-05-74 (1) to TtSlyDcys-Her3 (SEQ ID NO:
18) in Comparison with Anti-HER3 Antibody MOR09823 (2) Described in
WO2012/22814.
A Biacore.TM. T200 instrument (GE Healthcare) was mounted with CM5
series sensor and was normalized in HBS-ET+ buffer (10 mM HEPES pH
7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w/v Tween 20) according to the
manufacturer's instructions. The sample buffer was the system
buffer supplemented with 1 mg/ml CMD (Carboxymethyldextran). The
system operated at 37.degree. C. A double antibody capture system
was established on the sensor surface. 6500 RU mAb<M-IgG>R
was immobilized according to the manufacturer's instructions using
EDC/NHS chemistry on all flow cells. The sensor was deactivated
using 1M ethanolamine. Flow cell 1 served as a reference and was
captured for 1 min at 10 .mu.l/min with anti-TSH IgG1 antibody. On
flow cell 2 M-5-74 was captured for 1 min at 10 .mu.l/min. On flow
cell 3 a murine anti-human FC pan antibody was captured 1 min at 10
.mu.l/min followed by the injection of the anti-HER3 antibody
M-05-74 (1) or of anti-HER3 antibody MOR09823 antibody for 1 min at
10 .mu.l/min. The flow rate was set to 60 .mu.l/min. The analyte in
solution TtSlyDcys-HER3 (SEQ ID NO: 18) was injected at
concentrations of 0 nM and 150 nM for 5 min and the dissociation
was monitored for 600 sec. The sensor was fully regenerated by one
injection at 10 .mu.l/min for 3 min with 10 mM glycine pH 1.7
buffer.
FIG. 20 depicts a sensorgram overlay plot showing binding signals
at 150 nM of, TtSlyDcys-Her3 and buffer. The overlay plot above
shows the antibody M-5-74 binding at 150 nM TtSlyDcys-Her3 (1).
MOR09823 antibody does not bind TtSlyDcas-Her3 (2). (3) shows the
background binding signal of the TtSlyDcas-HER3 versus the
mAb<M-IgG>R capture surface. The anti-HER3 antibody MOR09823
(2) described in WO2012/22814 does not show any interaction at 150
nM TtSlyDcys-Her3. The positive control antibody M-05-74 (1) shows
significiant binding versus TtSlyDcas-Her3. No interaction could be
determined with both antibodies when injecting 150 nM TtSlyDcys (no
HER-3 insertion) (data not shown).
Example 15
Generation and Evaluation of HER3/HER2 Bispecific Antibody
DIB.times.PERT Binding to the Beta-Hairpin of HER3 and Domain II of
HER2
Material and Methods
Recombinant DNA Techniques
Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
Gene Synthesis
Desired gene synthesis fragments were ordered according to given
specifications at Geneart (Regensburg, Germany).
The 600-1500 bp long gene segments, which were flanked by singular
restriction endonuclease cleavage sites, were cloned via the
indicated restriction sites into a pUC expression vector, e.g.
BamHI/XbaI, BamHI/XhoI (FIGS. 22-25). The DNA sequences of the
subcloned gene fragments were confirmed by DNA sequencing.
DNA Sequence Determination
DNA sequences were determined by double strand sequencing performed
at Sequiserve GmbH (Vaterstetten, Germany).
DNA and Protein Sequence Analysis and Sequence Data Management
Infomax's Vector NT1 Advance.RTM. suite version 11.5.0 was used for
sequence creation, mapping, analysis, annotation and
illustration.
CrossMab Design
The CrossMab technology (Schaefer et al. 2011) combines two heavy
and light chains of different parental antibodies with different
specificities into one IgG-like format. To facilitate
heterodimerization of two different heavy chains, the
`knob-into-hole` technology is applied, whereby one smaller amino
acid is exchanged by a larger amino acid in one CH3 domain
(`knob`). In the CH3 domain of the second antibody, a larger amino
acid is exchanged by smaller amino acids (`hole`). To ensure the
correct assimilation of light chains with corresponding heavy
chains, the CH1 domain of one heavy chain is exchanged with the
CKappa (CK) domain of the respective light chain. The end product
in general is called CrossMab. Here, the M-05-74 (DIB-74) antibody
light (FIG. 22) and heavy chain (FIG. 23) were used, whereby a
`knob` mutation was introduced into the CH3 domain of the DIB heavy
chain. As a second parental antibody Pertuzumab was used. Here, the
crossing-over was induced between the CK domain of the light chain
(FIG. 24) and the CH1 domain of the heavy chain (FIG. 25). In the
CH3 domain `hole` mutations were introduced. The resulting CrossMab
is called DIB.times.PERT (see sequences SEQ ID NOs 68-71, whereby
the x in front of PERT (Pertuzumab) indicates, that the cross-over
was introduced in the Pertuzumab site (FIG. 26).
Expression of DIB.times.PERT
For the expression of DIB.times.PERT by HEK293F cells, plasmid DNA
was obtain by QIAGEN Plasmid Plus Maxi Kit (Qiagen, Hilden,
Germany), according to the manufacturer's indications. Cells were
seeded with 1.0E+06 cells per ml Gibco.RTM. FreeStyle.TM. 293
Expression Medium. One liter of cells was transfected with 22 pmol
of each of the four plasmids (CB01_DIB-LC_VL-CK,
CB02_DIB-HC_VH-CH1-CH2-CH3_knob, NN21 pUC-Exp_xMab_Pertuzu_LC, NN24
pUC-xPertuzu-SSKHC2-RSE), using the 293-Free.TM. Transfection
Reagent, according to the manufacturer's instructions. Cells were
incubated for seven days at 37.degree. C., 8% CO2 and 80% air
humidity, shaking at 150 rpm (LabTherm LT-XC, Kuhner AG,
Birsfelden, Schweiz). After incubation, 50 mM PMSF (Sigma-Aldrich,
Steinheim, Germany), 1 ng MgCl2 (Merck GmbH, Darmstadt, Germany)
and 10 U/ml DNaseI (Roche Diagnostics GmbH, Mannheim, Germany) were
added and cells were incubated for 30 minutes. The antibody
containing supernatant was harvested by pelleting the cells for 30
minutes at 890.times.g (Rotanta 460R, Andreas Hettich GmbH,
Tuttlingen, Germany). Until purification, the supernatant was
stored at -20.degree. C.
Purification of DIB.times.PERT
The antibody was isolated using an AktaAvant instrument (GE
Healthcare, Munchen, Germany). A protein A HiTrap MabSelect
SuRe.TM. (5 ml) (GE Healthcare, Munchen, Germany) was equilibrated
with 50 mM KH.sub.2PO.sub.4, 150 mM KCl, pH 7.4 system buffer. The
supernatant was filtered with 0.22 .mu.m sterile filter units
beforehand and then applied onto the column with a flow rate of 0.9
ml/min overnight. Subsequently, unbound material was removed, using
the system buffer at a flow rate of 2 ml/min. Then, bound
antibodies were eluted from the column using 0.1 M Na-Citrate pH
3.7 at a flow rate of 1 ml/min and directly neutralized with 1 M
L-Arginine buffer. Desired fractions were pooled and purified by
gel permeation chromatography (GPC), thereby dialyzing the buffer
into 20 mM Histidine, 140 mM NaCl, pH 6.0 storage buffer. The
DIB.times.PERT end-product quantity was analyzed by spectroscopy at
280 nm. The quality was controlled by GPC, using a TSK-Gel.RTM.
QC-PAK GF30 column (Tosoh Bioscience GmbH, Stuttgart, Germany) and
a UltiMate.TM. 3000 Dionex instrument (Fisher Scientific GmbH,
Schwerte, Germany) (FIGS. 27 A and B). As a second quality control
a 4-12% Bis-Tris SDS-PAGE (Life Technologies GmbH, Darmstadt,
Germany) was used, with reducing and non-reducing conditions (FIG.
27 C). By reducing conditions the covalent disulfidbridges between
heavy and light chains are disrupted.
To assess the purity and aggregation state of DIB.times.PERT, an
analytical GPC was conducted, using a TSK-Gel.RTM. QC-PAK GF30
column (Tosoh Bioscience, Stuttgart, Germany). DIB.times.PERT was
purified with a relative GPC peak area of 96% (FIG. 27 A).
Comparison with the standard curve reference confirmed a molar mass
of 145 kDa. The SDS-PAGE (FIG. 27 B) revealed a protein band at
approximately 145 kDa under non-reducing condition. Under
reducing-condition the disulfide bonds between heavy and light
chains are disrupting. Therefore, two bands were found in the
SDS-PAGE, which could be assigned to the heavy (approx. 50 kDa) and
light (approx. 25 kDa) antibody chains (FIG. 27 C). The purity of
the DIB.times.PERT end product was adequate for subsequent
experiments and analyses.
Example 16
Determination of HER3/HER2 Bispecific Antibody DIB.times.PERT
Kinetic Features by SPR Analyses
In its equilibrium state, the HER3-ECD is in its "closed
confirmation", which does mean, the heterodimerization HER3
.beta.-hairpin motive is tethered via non-covalent interactions to
the HER3-ECD domain IV. It is supposed, that the "closed" HER3
conformation can be opened via the binding of the ligand heregulin
at a specific HER3 heregulin binding site. This takes place at the
HER3 interface formed by the HER3-ECD domains I and domain III. By
this interaction it is believed, that the HER3 receptor is
activated and transferred into its "open conformation". When this
occurs, the HER3 .beta.-hairpin is accessible for the described
antibodies. This mode of action can be simulated in vitro by a
Biacore.TM. experiment.
To investigate, if the kinetic features of parental antibodies
DIB-74 and Pertuzumab were retained by DIB.times.PERT, real-time
data were collected, using SPR analyses, a Biacore.TM. B3000
instrument (GE Healthcare) was used to kinetically assess the
monoclonal antibodies at 25.degree. C. for their behavior to the
heregulin-activated HER3 Extracellular Domain (HER3-ECD) and the
constitutive open HER2-ECD. A CM5 series sensor was mounted into
the system and was normalized in HBS-ET buffer (10 mM HEPES pH 7.4,
150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween 20) according to the
manufacturer's instructions. The sample buffer was the system
buffer supplemented with 1 mg/ml CMD (Carboxymethyldextran, Sigma
#86524). The system operated at 25.degree. C. 10000 RU monoclonal
murine anti-human Fc antibody (MAK<h-Fc>M-R10Z8E9, Roche
Diagnostics GmbH, Penzberg, Germany) were immobilized using EDC/NHS
chemistry on all four flow cells. The sensor was deactivated using
1M ethanolamine.
The analytes in solution tested were 270 nM human recombinant
HER2-ECD (69.6 kDa) and 270 nM human recombinant HER3-ECD (68 kDa)
which was incubated with a 3-fold molar excess of human
Heregulin1.beta. (HRG1.beta.) a 44 kDa homodimeric protein, for 60
min at room temperature resulting in HER3-ECD/HRG1.beta. complex.
Analytes in solution were injected at different concentration steps
of 0 nM, 3.3 nM, 10 nM, 30 nM, 90 nM and 270 nM for 5 min at a flow
rate of 30 .mu.l/min (FIG. 28). The dissociation was monitored for
10 min. Kinetic signatures were evaluated, where possible,
according to a Langmuir fit.
For assessing the simultaneous binding capacity of DIB.times.PERT
to both HER2-ECD and the HER3-ECD/HRG1.beta. complex, a second
assay setup was used. Herein, the analytes HER2-ECD (270 nM) and
HER3-ECD/HRG1.beta. complex (270 nM HER3-ECD with a threefold
surplus of HRG1.beta.) were injected subsequently, or vice versa
(FIG. 29). The association and dissociation rates were monitored
for 10 minutes and 8 minutes, respectively.
DIB.times.PERT Retained the Specificity of the Parental
Antibodies
DIB.times.PERT and the parental antibodies DIB-74 in the monovalent
MoAb format and Pertuzumab in the bivalent IgG format, were
compared, using SPR analyses. DIB.times.PERT retained the
specificities of its parental antibodies Pertuzumab and DIB-74 and
bound to the HER2-ECD as well as the HER3-ECD/HRG1.beta. complex
(FIG. 7). The data show, that the affinity of Pertuzumab for the
HER2-ECD (K.sub.D 1.7 nM) was retained in DIB.times.PERT (K.sub.D
1.6 nM). The Molar Ratio of Pertuzumab (MR=1.3) was two-fold higher
than of DIB.times.PERT (MR=0.6 nM). The ability to bind the open
HER3-ECD (HER3-ECD/HRG1.beta.) was also retained in DIB.times.PERT.
The affinity of DIB.times.PERT to the HER3-ECD/HRG1.beta. complex
(K.sub.D 3.9 nM) was comparable to that of the DIB-MoAb to the
HER3-ECD/HRG1.beta. complex (K.sub.D 2.1 nM). Both antibodies bound
with a substoichiometric molar ratio of 0.6 and 0.5, respectively
(Table 12).
TABLE-US-00027 TABLE 12 Kinetic parameters of DIBxPERT and the
parental antibodies DIB-MoAb and Pertuzumab, determined by SPR
analyses, using a B3000 Biacore instrument (Ge Healthcare). CL
R.sub.max k.sub.a k.sub.d t.sub.1/2-diss K.sub.D Ligand Analyte
(RU) (RU) (1/Ms) (1/s) (min) (nM) MR PERT HER2-ECD 160 99 6.9E+04
1.2E-04 100 1.7 1.3 DIBxPERT HER2-ECD 165 46 8.5E+04 1.4E-04 83 1.6
0.6 DIB-MoAb HER3- 121 81 1.1E+05 2.3E-04 51 2.1 0.6 ECD/HRG1.beta.
DIBxPERT HER3- 162 64 8.4E+04 3.3E-04 35 3.9 0.5 ECD/HRG1.beta. CL:
Capture level in Response Units, R.sub.max: maximal binding level
of the analytes, k.sub.a: association rate constant in 1/Ms,
k.sub.d: dissociation rate constant in 1/s, t.sub.1/2-diss: complex
half-life period in minutes, K.sub.D: equilibrium dissociation
constant, MR: molar ratio.
Simultaneous Binding of DIB.times.PERT to Soluble HER2-ECD and
HER3-ECD/HRG1.beta. Complex
The simultaneous binding capacity of DIB.times.PERT to the soluble
HER2-ECD and HER3-ECD/HRG1.beta. complex was assessed using SPR
analyses (FIG. 29). We found, that DIB.times.PERT was able to bind
the HER3-ECD/HRG1.beta. complex with one valence (MR=0.7), even
when already bound to the HER2-ECD with the second valence
(MR=0.8). Reciprocal, DIB.times.PERT bound the HER2-ECD with one
valence (MR=0.8), when already bound to the HER3-ECD/HRG1.beta.
complex with the second valence (MR=0.8). The data show, that
DIB.times.PERT is able to bind both targets (HER2-ECD and
HER3-ECD/HRG1.beta.) at the same time, when using soluble
analytes.
Example 17
Inhibition of Tumor Cell Proliferation of HER3/HER2 Bispecific
Antibody DIB.times.PERT in MDA-MB-175 VII Cells
The anti-tumor efficacy of DIB.times.PERT was assessed in a cell
proliferation assay, using MDA-MB-175 cells (VII Human Breast
Carcinoma Cells, ATCC catalog no. HTB-25). 20,000 cells per well
were seeded into sterile 96 well tissue culture plates with
DMEM/F12 cell culture medium, containing 10% FCS and 2 mM
L-Glutamine and incubated at 37.degree. C. with 5% CO.sub.2 for one
day. The cells are slow growing cells with a doubling time of
approximately 3 days. Cells were starved with 0.5% FCS containing
DMEM/F12 cell culture medium, containing 2 mM L-Glutamine.
DIB.times.PERT and control antibodies were added in dilution series
and further incubated for 6 days. The applied antibodies are listed
in table 13. Cell viability was then assessed using the
alamarBlue.RTM. readout. EC.sub.50 values were calculated using
means of triplicates for each antibody concentration (FIG. 30).
TABLE-US-00028 TABLE 13 Antibodies used for the inhibition of tumor
cell proliferation in MDA-MB-175 VII cells in vitro. Additionally
to the below mentioned single treatments, the combination
treatments of DIB-74 with Pertuzumab and RG7116 with Pertuzumab
were applied in vitro. Antibody Format Valence Specificity DIBxPERT
CrossMab bivalent anti-HER2 subdomain II anti-HER3 .beta.-hairpin
DIB-MoAb MoAb monovalent anti-HER3 .beta.-hairpin (monovalent
antibody based on M-05-74) DIB-74 (M-05- Monoclonal bivalent
anti-HER3 .beta.-hairpin 74) IgG Pertuzumab Monoclonal bivalent
anti-HER2 subdomain IgG II RG7116 Monoclonal bivalent anti-HER3
subdomain I (<HER3> Mab IgG binding to domain I) Isotype
control Polyclonal IgG bivalent no specific target
Inhibition of Tumor Cell Proliferation of DIB.times.PERT in
MDA-MB-175 VII Cells
The inhibition of tumor cell proliferation of DIB.times.PERT was
examined in vitro, using MDA-MB-175 VII cells. In the MDA-MB-175
VII cell line (doubling time 3 days) the oncogenic signal arises
from an autocrine HRG growth loop. Cells were incubated with the
series diluted antibodies DIB.times.PERT, DIB-MoAb, DIB-74,
Pertuzumab, RG7116, the combinations of DIB-74 and Pertuzumab and
of RG7116 and Pertuzumab and an Isotype control (FIG. 30). After 6
days, the maximal growth inhibition of 79% was achieved by
DIB.times.PERT, in contrast to the other mono and combination
treatments (Table 14). The second highest maximum inhibitory effect
was seen with the combinations of DIB-74 and Pertuzumab (76%) and
RG7116 and Pertuzumab (76%) treated cells. The EC.sub.50 for
DIB.times.PERT-mediated growth inhibition was 1 nM and thereby
superior to the EC.sub.50 of control antibodies. Pertuzumab
mono-treatment or Pertuzumab in combination with DIB-74 or RG7116
showed growth inhibition EC.sub.50 of 2 nM. Compared to that,
RG7116 (EC.sub.50 7 nM) and DIB (EC.sub.50 26 nM) alone mediated a
lower tumor growth inhibition in vitro.
TABLE-US-00029 TABLE 14 Inhibition of tumor cell proliferation of
DIBxPERT in MDA-MB-175 VII cells. Tumor cell proliferation
inhibition (%) EC50 Antibody minimum maximum (nM) DIBxPERT -14 79 1
DIB-MoAb -12 n/a n/a monovalent M- 05-74 DIB (M-05-74) -15 56 26
PERT -12 67 2 RG7116 -17 58 7 Combination of -15 76 2 DIB and PERT
Combination -16 76 2 RG7116 and PERT Isotype control -19 -13 n/a
DIB: murine DIB-74 IgG; PERT: Pertuzumab IgG; RG7116: humanized
anti-HER3 subdomain I IgG; Isotype control: polyclonal human
antibody; n/a: not applicable.
SEQUENCE LISTINGS
1
71119PRTHomo Sapiens 1Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe
Gln Leu Glu Pro Asn1 5 10 15Pro His Thr219PRTHomo Sapiens 2Pro Gln
Thr Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His Asn1 5 10 15Phe
Asn Ala31323PRTHomo Sapiens 3Ser Glu Val Gly Asn Ser Gln Ala Val
Cys Pro Gly Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala Glu
Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30Leu Tyr Glu Arg Cys Glu Val
Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45Thr Gly His Asn Ala Asp
Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60Thr Gly Tyr Val Leu
Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn Leu
Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90 95Ala Ile
Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu 100 105
110Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val
115 120 125Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile
Asp Trp 130 135 140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val
Val Lys Asp Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His Glu
Val Cys Lys Gly Arg Cys Trp 165 170 175Gly Pro Gly Ser Glu Asp Cys
Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190Pro Gln Cys Asn Gly
His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205His Asp Glu
Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215 220Phe
Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys225 230
235 240Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro
Asn 245 250 255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala
Ser Cys Pro 260 265 270His Asn Phe Val Val Asp Gln Thr Ser Cys Val
Arg Ala Cys Pro Pro 275 280 285Asp Lys Met Glu Val Asp Lys Asn Gly
Leu Lys Met Cys Glu Pro Cys 290 295 300Gly Gly Leu Cys Pro Lys Ala
Cys Glu Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe Gln Thr Val
Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr 325 330 335Lys Ile
Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp 340 345
350Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe
355 360 365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser
Trp Pro 370 375 380Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu
Thr Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn Arg Gly Phe
Ser Leu Leu Ile Met Lys Asn 405 410 415Leu Asn Val Thr Ser Leu Gly
Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430Gly Arg Ile Tyr Ile
Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435 440 445Leu Asn Trp
Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp 450 455 460Ile
Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val465 470
475 480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro
Gly 485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val
Cys Val Thr 500 505 510His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu
Phe Ala His Glu Ala 515 520 525Glu Cys Phe Ser Cys His Pro Glu Cys
Gln Pro Met Glu Gly Thr Ala 530 535 540Thr Cys Asn Gly Ser Gly Ser
Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555 560Arg Asp Gly Pro
His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly 565 570 575Ala Lys
Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg 580 585
590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln
595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His
Leu Thr 610 615 620Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile
Phe Met Met Leu625 630 635 640Gly Gly Thr Phe Leu Tyr Trp Arg Gly
Arg Arg Ile Gln Asn Lys Arg 645 650 655Ala Met Arg Arg Tyr Leu Glu
Arg Gly Glu Ser Ile Glu Pro Leu Asp 660 665 670Pro Ser Glu Lys Ala
Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr 675 680 685Glu Leu Arg
Lys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val 690 695 700His
Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val705 710
715 720Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln
Ala 725 730 735Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His
Ala His Ile 740 745 750Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser
Leu Gln Leu Val Thr 755 760 765Gln Tyr Leu Pro Leu Gly Ser Leu Leu
Asp His Val Arg Gln His Arg 770 775 780Gly Ala Leu Gly Pro Gln Leu
Leu Leu Asn Trp Gly Val Gln Ile Ala785 790 795 800Lys Gly Met Tyr
Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu 805 810 815Ala Ala
Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala 820 825
830Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu
835 840 845Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu
Ser Ile 850 855 860His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp
Ser Tyr Gly Val865 870 875 880Thr Val Trp Glu Leu Met Thr Phe Gly
Ala Glu Pro Tyr Ala Gly Leu 885 890 895Arg Leu Ala Glu Val Pro Asp
Leu Leu Glu Lys Gly Glu Arg Leu Ala 900 905 910Gln Pro Gln Ile Cys
Thr Ile Asp Val Tyr Met Val Met Val Lys Cys 915 920 925Trp Met Ile
Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn 930 935 940Glu
Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys945 950
955 960Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly
Leu 965 970 975Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu
Leu Asp Leu 980 985 990Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu
Ala Thr Thr Thr Leu 995 1000 1005Gly Ser Ala Leu Ser Leu Pro Val
Gly Thr Leu Asn Arg Pro Arg 1010 1015 1020Gly Ser Gln Ser Leu Leu
Ser Pro Ser Ser Gly Tyr Met Pro Met 1025 1030 1035Asn Gln Gly Asn
Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser 1040 1045 1050Gly Ser
Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met 1055 1060
1065Pro Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr
1070 1075 1080Gly Ser Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys
Arg Ser 1085 1090 1095Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly
Asp Ser Ala Tyr 1100 1105 1110His Ser Gln Arg His Ser Leu Leu Thr
Pro Val Thr Pro Leu Ser 1115 1120 1125Pro Pro Gly Leu Glu Glu Glu
Asp Val Asn Gly Tyr Val Met Pro 1130 1135 1140Asp Thr His Leu Lys
Gly Thr Pro Ser Ser Arg Glu Gly Thr Leu 1145 1150 1155Ser Ser Val
Gly Leu Ser Ser Val Leu Gly Thr Glu Glu Glu Asp 1160 1165 1170Glu
Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg His Ser 1175 1180
1185Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly Tyr
1190 1195 1200Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu
Gly Ser 1205 1210 1215Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile
Met Pro Thr Ala 1220 1225 1230Gly Thr Thr Pro Asp Glu Asp Tyr Glu
Tyr Met Asn Arg Gln Arg 1235 1240 1245Asp Gly Gly Gly Pro Gly Gly
Asp Tyr Ala Ala Met Gly Ala Cys 1250 1255 1260Pro Ala Ser Glu Gln
Gly Tyr Glu Glu Met Arg Ala Phe Gln Gly 1265 1270 1275Pro Gly His
Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr 1280 1285 1290Leu
Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp 1295 1300
1305Tyr Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr
1310 1315 13204624PRTHomo Sapiens 4Ser Glu Val Gly Asn Ser Gln Ala
Val Cys Pro Gly Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala
Glu Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30Leu Tyr Glu Arg Cys Glu
Val Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45Thr Gly His Asn Ala
Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60Thr Gly Tyr Val
Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn
Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90 95Ala
Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu 100 105
110Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val
115 120 125Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile
Asp Trp 130 135 140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val
Val Lys Asp Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His Glu
Val Cys Lys Gly Arg Cys Trp 165 170 175Gly Pro Gly Ser Glu Asp Cys
Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190Pro Gln Cys Asn Gly
His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205His Asp Glu
Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215 220Phe
Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys225 230
235 240Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro
Asn 245 250 255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala
Ser Cys Pro 260 265 270His Asn Phe Val Val Asp Gln Thr Ser Cys Val
Arg Ala Cys Pro Pro 275 280 285Asp Lys Met Glu Val Asp Lys Asn Gly
Leu Lys Met Cys Glu Pro Cys 290 295 300Gly Gly Leu Cys Pro Lys Ala
Cys Glu Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe Gln Thr Val
Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr 325 330 335Lys Ile
Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp 340 345
350Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe
355 360 365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser
Trp Pro 370 375 380Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu
Thr Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn Arg Gly Phe
Ser Leu Leu Ile Met Lys Asn 405 410 415Leu Asn Val Thr Ser Leu Gly
Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430Gly Arg Ile Tyr Ile
Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435 440 445Leu Asn Trp
Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp 450 455 460Ile
Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val465 470
475 480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro
Gly 485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val
Cys Val Thr 500 505 510His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu
Phe Ala His Glu Ala 515 520 525Glu Cys Phe Ser Cys His Pro Glu Cys
Gln Pro Met Glu Gly Thr Ala 530 535 540Thr Cys Asn Gly Ser Gly Ser
Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555 560Arg Asp Gly Pro
His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly 565 570 575Ala Lys
Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg 580 585
590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln
595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His
Leu Thr 610 615 62051283PRTHomo Sapiens 5Gln Ser Val Cys Ala Gly
Thr Glu Asn Lys Leu Ser Ser Leu Ser Asp1 5 10 15Leu Glu Gln Gln Tyr
Arg Ala Leu Arg Lys Tyr Tyr Glu Asn Cys Glu 20 25 30Val Val Met Gly
Asn Leu Glu Ile Thr Ser Ile Glu His Asn Arg Asp 35 40 45Leu Ser Phe
Leu Arg Ser Val Arg Glu Val Thr Gly Tyr Val Leu Val 50 55 60Ala Leu
Asn Gln Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg Ile Ile65 70 75
80Arg Gly Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile Phe Leu
85 90 95Asn Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly Leu
Lys 100 105 110Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp
Gln Asn Lys 115 120 125Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln
Asp Ile Val Arg Asn 130 135 140Pro Trp Pro Ser Asn Leu Thr Leu Val
Ser Thr Asn Gly Ser Ser Gly145 150 155 160Cys Gly Arg Cys His Lys
Ser Cys Thr Gly Arg Cys Trp Gly Pro Thr 165 170 175Glu Asn His Cys
Gln Thr Leu Thr Arg Thr Val Cys Ala Glu Gln Cys 180 185 190Asp Gly
Arg Cys Tyr Gly Pro Tyr Val Ser Asp Cys Cys His Arg Glu 195 200
205Cys Ala Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe Ala Cys
210 215 220Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro
Gln Thr225 230 235 240Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu
His Asn Phe Asn Ala 245 250 255Lys Tyr Thr Tyr Gly Ala Phe Cys Val
Lys Lys Cys Pro His Asn Phe 260 265 270Val Val Asp Ser Ser Ser Cys
Val Arg Ala Cys Pro Ser Ser Lys Met 275 280 285Glu Val Glu Glu Asn
Gly Ile Lys Met Cys Lys Pro Cys Thr Asp Ile 290 295 300Cys Pro Lys
Ala Cys Asp Gly Ile Gly Thr Gly Ser Leu Met Ser Ala305 310 315
320Gln Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys Thr Lys
325 330 335Ile Asn Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly
Asp Pro 340 345 350Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu
Asn Val Phe Arg 355 360 365Thr Val Arg Glu Ile Thr Gly Phe Leu Asn
Ile Gln Ser Trp Pro Pro 370 375 380Asn Met Thr Asp Phe Ser Val Phe
Ser Asn Leu Val Thr Ile Gly Gly385 390 395 400Arg Val Leu Tyr Ser
Gly Leu Ser Leu Leu Ile Leu Lys Gln Gln Gly 405 410 415Ile Thr Ser
Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala Gly Asn 420 425 430Ile
Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His Thr Ile Asn 435 440
445Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile Arg Asp
450 455 460Asn Arg Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys
Asn His465 470
475 480Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln Cys
Leu 485 490 495Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu
Ser Cys Asn 500 505 510Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn
Gly Ser Ile Cys Val 515 520 525Glu Cys Asp Pro Gln Cys Glu Lys Met
Glu Asp Gly Leu Leu Thr Cys 530 535 540His Gly Pro Gly Pro Asp Asn
Cys Thr Lys Cys Ser His Phe Lys Asp545 550 555 560Gly Pro Asn Cys
Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn 565 570 575Ser Phe
Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys His Pro Cys 580 585
590His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys
595 600 605Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His
Ala Arg 610 615 620Thr Pro Leu Ile Ala Ala Gly Val Ile Gly Gly Leu
Phe Ile Leu Val625 630 635 640Ile Val Gly Leu Thr Phe Ala Val Tyr
Val Arg Arg Lys Ser Ile Lys 645 650 655Lys Lys Arg Ala Leu Arg Arg
Phe Leu Glu Thr Glu Leu Val Glu Pro 660 665 670Leu Thr Pro Ser Gly
Thr Ala Pro Asn Gln Ala Gln Leu Arg Ile Leu 675 680 685Lys Glu Thr
Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly Ala Phe 690 695 700Gly
Thr Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr Val Lys705 710
715 720Ile Pro Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro Lys
Ala 725 730 735Asn Val Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser
Met Asp His 740 745 750Pro His Leu Val Arg Leu Leu Gly Val Cys Leu
Ser Pro Thr Ile Gln 755 760 765Leu Val Thr Gln Leu Met Pro His Gly
Cys Leu Leu Glu Tyr Val His 770 775 780Glu His Lys Asp Asn Ile Gly
Ser Gln Leu Leu Leu Asn Trp Cys Val785 790 795 800Gln Ile Ala Lys
Gly Met Met Tyr Leu Glu Glu Arg Arg Leu Val His 805 810 815Arg Asp
Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val 820 825
830Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys
835 840 845Glu Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met
Ala Leu 850 855 860Glu Cys Ile His Tyr Arg Lys Phe Thr His Gln Ser
Asp Val Trp Ser865 870 875 880Tyr Gly Val Thr Ile Trp Glu Leu Met
Thr Phe Gly Gly Lys Pro Tyr 885 890 895Asp Gly Ile Pro Thr Arg Glu
Ile Pro Asp Leu Leu Glu Lys Gly Glu 900 905 910Arg Leu Pro Gln Pro
Pro Ile Cys Thr Ile Asp Val Tyr Met Val Met 915 920 925Val Lys Cys
Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Lys Glu 930 935 940Leu
Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu945 950
955 960Val Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn Asp
Ser 965 970 975Lys Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu
Asp Met Met 980 985 990Asp Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe
Asn Ile Pro Pro Pro 995 1000 1005Ile Tyr Thr Ser Arg Ala Arg Ile
Asp Ser Asn Arg Ser Glu Ile 1010 1015 1020Gly His Ser Pro Pro Pro
Ala Tyr Thr Pro Met Ser Gly Asn Gln 1025 1030 1035Phe Val Tyr Arg
Asp Gly Gly Phe Ala Ala Glu Gln Gly Val Ser 1040 1045 1050Val Pro
Tyr Arg Ala Pro Thr Ser Thr Ile Pro Glu Ala Pro Val 1055 1060
1065Ala Gln Gly Ala Thr Ala Glu Ile Phe Asp Asp Ser Cys Cys Asn
1070 1075 1080Gly Thr Leu Arg Lys Pro Val Ala Pro His Val Gln Glu
Asp Ser 1085 1090 1095Ser Thr Gln Arg Tyr Ser Ala Asp Pro Thr Val
Phe Ala Pro Glu 1100 1105 1110Arg Ser Pro Arg Gly Glu Leu Asp Glu
Glu Gly Tyr Met Thr Pro 1115 1120 1125Met Arg Asp Lys Pro Lys Gln
Glu Tyr Leu Asn Pro Val Glu Glu 1130 1135 1140Asn Pro Phe Val Ser
Arg Arg Lys Asn Gly Asp Leu Gln Ala Leu 1145 1150 1155Asp Asn Pro
Glu Tyr His Asn Ala Ser Asn Gly Pro Pro Lys Ala 1160 1165 1170Glu
Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn Thr Phe Ala 1175 1180
1185Asn Thr Leu Gly Lys Ala Glu Tyr Leu Lys Asn Asn Ile Leu Ser
1190 1195 1200Met Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp
Tyr Trp 1205 1210 1215Asn His Ser Leu Pro Pro Arg Ser Thr Leu Gln
His Pro Asp Tyr 1220 1225 1230Leu Gln Glu Tyr Ser Thr Lys Tyr Phe
Tyr Lys Gln Asn Gly Arg 1235 1240 1245Ile Arg Pro Ile Val Ala Glu
Asn Pro Glu Tyr Leu Ser Glu Phe 1250 1255 1260Ser Leu Lys Pro Gly
Thr Val Leu Pro Pro Pro Pro Tyr Arg His 1265 1270 1275Arg Asn Thr
Val Val 12806626PRTHomo Sapiens 6Gln Ser Val Cys Ala Gly Thr Glu
Asn Lys Leu Ser Ser Leu Ser Asp1 5 10 15Leu Glu Gln Gln Tyr Arg Ala
Leu Arg Lys Tyr Tyr Glu Asn Cys Glu 20 25 30Val Val Met Gly Asn Leu
Glu Ile Thr Ser Ile Glu His Asn Arg Asp 35 40 45Leu Ser Phe Leu Arg
Ser Val Arg Glu Val Thr Gly Tyr Val Leu Val 50 55 60Ala Leu Asn Gln
Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg Ile Ile65 70 75 80Arg Gly
Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile Phe Leu 85 90 95Asn
Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly Leu Lys 100 105
110Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln Asn Lys
115 120 125Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val
Arg Asn 130 135 140Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn
Gly Ser Ser Gly145 150 155 160Cys Gly Arg Cys His Lys Ser Cys Thr
Gly Arg Cys Trp Gly Pro Thr 165 170 175Glu Asn His Cys Gln Thr Leu
Thr Arg Thr Val Cys Ala Glu Gln Cys 180 185 190Asp Gly Arg Cys Tyr
Gly Pro Tyr Val Ser Asp Cys Cys His Arg Glu 195 200 205Cys Ala Gly
Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe Ala Cys 210 215 220Met
Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro Gln Thr225 230
235 240Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His Asn Phe Asn
Ala 245 250 255Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys Lys Cys Pro
His Asn Phe 260 265 270Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys
Pro Ser Ser Lys Met 275 280 285Glu Val Glu Glu Asn Gly Ile Lys Met
Cys Lys Pro Cys Thr Asp Ile 290 295 300Cys Pro Lys Ala Cys Asp Gly
Ile Gly Thr Gly Ser Leu Met Ser Ala305 310 315 320Gln Thr Val Asp
Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys Thr Lys 325 330 335Ile Asn
Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly Asp Pro 340 345
350Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn Val Phe Arg
355 360 365Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp
Pro Pro 370 375 380Asn Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val
Thr Ile Gly Gly385 390 395 400Arg Val Leu Tyr Ser Gly Leu Ser Leu
Leu Ile Leu Lys Gln Gln Gly 405 410 415Ile Thr Ser Leu Gln Phe Gln
Ser Leu Lys Glu Ile Ser Ala Gly Asn 420 425 430Ile Tyr Ile Thr Asp
Asn Ser Asn Leu Cys Tyr Tyr His Thr Ile Asn 435 440 445Trp Thr Thr
Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile Arg Asp 450 455 460Asn
Arg Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys Asn His465 470
475 480Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln Cys
Leu 485 490 495Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu
Ser Cys Asn 500 505 510Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn
Gly Ser Ile Cys Val 515 520 525Glu Cys Asp Pro Gln Cys Glu Lys Met
Glu Asp Gly Leu Leu Thr Cys 530 535 540His Gly Pro Gly Pro Asp Asn
Cys Thr Lys Cys Ser His Phe Lys Asp545 550 555 560Gly Pro Asn Cys
Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn 565 570 575Ser Phe
Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys His Pro Cys 580 585
590His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys
595 600 605Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His
Ala Arg 610 615 620Thr Pro62571186PRTHomo Sapiens 7Leu Glu Glu Lys
Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr
Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30Asn Cys
Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45Asn
Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55
60Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu65
70 75 80Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu
Ala 85 90 95Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu
Leu Pro 100 105 110Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val
Arg Phe Ser Asn 115 120 125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile
Gln Trp Arg Asp Ile Val 130 135 140Ser Ser Asp Phe Leu Ser Asn Met
Ser Met Asp Phe Gln Asn His Leu145 150 155 160Gly Ser Cys Gln Lys
Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175Gly Ala Gly
Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190Gln
Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200
205His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys
210 215 220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp
Thr Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr
Gln Met Asp Val Asn 245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly Ala
Thr Cys Val Lys Lys Cys Pro 260 265 270Arg Asn Tyr Val Val Thr Asp
His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285Ala Asp Ser Tyr Glu
Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300Cys Glu Gly
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu305 310 315
320Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys
325 330 335Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val
Ala Phe 340 345 350Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
Pro Gln Glu Leu 355 360 365Asp Ile Leu Lys Thr Val Lys Glu Ile Thr
Gly Phe Leu Leu Ile Gln 370 375 380Ala Trp Pro Glu Asn Arg Thr Asp
Leu His Ala Phe Glu Asn Leu Glu385 390 395 400Ile Ile Arg Gly Arg
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415Val Ser Leu
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430Ser
Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440
445Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr
450 455 460Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr
Gly Gln465 470 475 480Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys
Trp Gly Pro Glu Pro 485 490 495Arg Asp Cys Val Ser Cys Arg Asn Val
Ser Arg Gly Arg Glu Cys Val 500 505 510Asp Lys Cys Asn Leu Leu Glu
Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525Ser Glu Cys Ile Gln
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540Ile Thr Cys
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His545 550 555
560Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met
565 570 575Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly
His Val 580 585 590Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
Thr Gly Pro Gly 595 600 605Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys
Ile Pro Ser Ile Ala Thr 610 615 620Gly Met Val Gly Ala Leu Leu Leu
Leu Leu Val Val Ala Leu Gly Ile625 630 635 640Gly Leu Phe Met Arg
Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg 645 650 655Arg Leu Leu
Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 660 665 670Glu
Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe 675 680
685Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
690 695 700Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val
Ala Ile705 710 715 720Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala
Asn Lys Glu Ile Leu 725 730 735Asp Glu Ala Tyr Val Met Ala Ser Val
Asp Asn Pro His Val Cys Arg 740 745 750Leu Leu Gly Ile Cys Leu Thr
Ser Thr Val Gln Leu Ile Thr Gln Leu 755 760 765Met Pro Phe Gly Cys
Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn 770 775 780Ile Gly Ser
Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly785 790 795
800Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala
805 810 815Arg Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr
Asp Phe 820 825 830Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu
Tyr His Ala Glu 835 840 845Gly Gly Lys Val Pro Ile Lys Trp Met Ala
Leu Glu Ser Ile Leu His 850 855 860Arg Ile Tyr Thr His Gln Ser Asp
Val Trp Ser Tyr Gly Val Thr Val865 870 875 880Trp Glu Leu Met Thr
Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala 885 890 895Ser Glu Ile
Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 900 905 910Pro
Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 915 920
925Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe
930 935 940Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln
Gly Asp945 950 955 960Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser
Asn Phe Tyr Arg Ala 965 970 975Leu Met Asp Glu Glu Asp Met Asp Asp
Val Val Asp Ala Asp Glu Tyr 980 985 990Leu Ile Pro Gln Gln Gly Phe
Phe Ser Ser Pro Ser Thr Ser Arg Thr 995 1000 1005Pro Leu Leu Ser
Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val 1010 1015 1020Ala Cys
Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu 1025
1030 1035Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly Ala
Leu 1040 1045 1050Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val
Pro Glu Tyr 1055 1060 1065Ile Asn Gln Ser Val Pro Lys Arg Pro Ala
Gly Ser Val Gln Asn 1070 1075 1080Pro Val Tyr His Asn Gln Pro Leu
Asn Pro Ala Pro Ser Arg Asp 1085 1090 1095Pro His Tyr Gln Asp Pro
His Ser Thr Ala Val Gly Asn Pro Glu 1100 1105 1110Tyr Leu Asn Thr
Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp 1115 1120 1125Ser Pro
Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu 1130 1135
1140Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys
1145 1150 1155Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala
Glu Tyr 1160 1165 1170Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile
Gly Ala 1175 1180 11858621PRTHomo Sapiens 8Leu Glu Glu Lys Lys Val
Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr Phe Glu
Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn 20 25 30Asn Cys Glu Val
Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg 35 40 45Asn Tyr Asp
Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr 50 55 60Val Leu
Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu65 70 75
80Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala
85 90 95Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu
Pro 100 105 110Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg
Phe Ser Asn 115 120 125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln
Trp Arg Asp Ile Val 130 135 140Ser Ser Asp Phe Leu Ser Asn Met Ser
Met Asp Phe Gln Asn His Leu145 150 155 160Gly Ser Cys Gln Lys Cys
Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp 165 170 175Gly Ala Gly Glu
Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala 180 185 190Gln Gln
Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys 195 200
205His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys
210 215 220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp
Thr Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr
Gln Met Asp Val Asn 245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly Ala
Thr Cys Val Lys Lys Cys Pro 260 265 270Arg Asn Tyr Val Val Thr Asp
His Gly Ser Cys Val Arg Ala Cys Gly 275 280 285Ala Asp Ser Tyr Glu
Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys 290 295 300Cys Glu Gly
Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu305 310 315
320Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys
325 330 335Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val
Ala Phe 340 345 350Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
Pro Gln Glu Leu 355 360 365Asp Ile Leu Lys Thr Val Lys Glu Ile Thr
Gly Phe Leu Leu Ile Gln 370 375 380Ala Trp Pro Glu Asn Arg Thr Asp
Leu His Ala Phe Glu Asn Leu Glu385 390 395 400Ile Ile Arg Gly Arg
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val 405 410 415Val Ser Leu
Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile 420 425 430Ser
Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala 435 440
445Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr
450 455 460Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr
Gly Gln465 470 475 480Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys
Trp Gly Pro Glu Pro 485 490 495Arg Asp Cys Val Ser Cys Arg Asn Val
Ser Arg Gly Arg Glu Cys Val 500 505 510Asp Lys Cys Asn Leu Leu Glu
Gly Glu Pro Arg Glu Phe Val Glu Asn 515 520 525Ser Glu Cys Ile Gln
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn 530 535 540Ile Thr Cys
Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His545 550 555
560Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met
565 570 575Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly
His Val 580 585 590Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
Thr Gly Pro Gly 595 600 605Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys
Ile Pro Ser 610 615 62091233PRTHomo Sapiens 9Thr Gln Val Cys Thr
Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser1 5 10 15Pro Glu Thr His
Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30Val Val Gln
Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45Leu Ser
Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60Ala
His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val65 70 75
80Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp
85 90 95Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser
Pro 100 105 110Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu
Ile Leu Lys 115 120 125Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu
Cys Tyr Gln Asp Thr 130 135 140Ile Leu Trp Lys Asp Ile Phe His Lys
Asn Asn Gln Leu Ala Leu Thr145 150 155 160Leu Ile Asp Thr Asn Arg
Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175Cys Lys Gly Ser
Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190Leu Thr
Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200
205Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly
210 215 220Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His
Ser Gly225 230 235 240Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr
Tyr Asn Thr Asp Thr 245 250 255Phe Glu Ser Met Pro Asn Pro Glu Gly
Arg Tyr Thr Phe Gly Ala Ser 260 265 270Cys Val Thr Ala Cys Pro Tyr
Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285Cys Thr Leu Val Cys
Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300Gly Thr Gln
Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys305 310 315
320Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser
325 330 335Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly
Ser Leu 340 345 350Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala
Ser Asn Thr Ala 355 360 365Pro Leu Gln Pro Glu Gln Leu Gln Val Phe
Glu Thr Leu Glu Glu Ile 370 375 380Thr Gly Tyr Leu Tyr Ile Ser Ala
Trp Pro Asp Ser Leu Pro Asp Leu385 390 395 400Ser Val Phe Gln Asn
Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415Gly Ala Tyr
Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430Leu
Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440
445Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe
450 455 460Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro
Glu Asp465 470 475 480Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln
Leu Cys Ala Arg Gly 485 490 495His Cys Trp Gly Pro Gly Pro Thr Gln
Cys Val Asn Cys Ser Gln Phe 500 505 510Leu Arg Gly Gln Glu Cys Val
Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525Pro Arg Glu Tyr Val
Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540Cys Gln Pro
Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp545 550 555
560Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala
565 570 575Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro
Ile Trp 580 585 590Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys
Pro Ile Asn Cys 595 600 605Thr His Ser Cys Val Asp Leu Asp Asp Lys
Gly Cys Pro Ala Glu Gln 610 615 620Arg Ala Ser Pro Leu Thr Ser Ile
Ile Ser Ala Val Val Gly Ile Leu625 630 635 640Leu Val Val Val Leu
Gly Val Val Phe Gly Ile Leu Ile Lys Arg Arg 645 650 655Gln Gln Lys
Ile Arg Lys Tyr Thr Met Arg Arg Leu Leu Gln Glu Thr 660 665 670Glu
Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met Pro Asn Gln Ala 675 680
685Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys Val Lys Val Leu
690 695 700Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile
Pro Asp705 710 715 720Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys
Val Leu Arg Glu Asn 725 730 735Thr Ser Pro Lys Ala Asn Lys Glu Ile
Leu Asp Glu Ala Tyr Val Met 740 745 750Ala Gly Val Gly Ser Pro Tyr
Val Ser Arg Leu Leu Gly Ile Cys Leu 755 760 765Thr Ser Thr Val Gln
Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu 770 775 780Leu Asp His
Val Arg Glu Asn Arg Gly Arg Leu Gly Ser Gln Asp Leu785 790 795
800Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Asp
805 810 815Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu
Val Lys 820 825 830Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu
Ala Arg Leu Leu 835 840 845Asp Ile Asp Glu Thr Glu Tyr His Ala Asp
Gly Gly Lys Val Pro Ile 850 855 860Lys Trp Met Ala Leu Glu Ser Ile
Leu Arg Arg Arg Phe Thr His Gln865 870 875 880Ser Asp Val Trp Ser
Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe 885 890 895Gly Ala Lys
Pro Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro Asp Leu 900 905 910Leu
Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp 915 920
925Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ser Glu Cys Arg
930 935 940Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala
Arg Asp945 950 955 960Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp
Leu Gly Pro Ala Ser 965 970 975Pro Leu Asp Ser Thr Phe Tyr Arg Ser
Leu Leu Glu Asp Asp Asp Met 980 985 990Gly Asp Leu Val Asp Ala Glu
Glu Tyr Leu Val Pro Gln Gln Gly Phe 995 1000 1005Phe Cys Pro Asp
Pro Ala Pro Gly Ala Gly Gly Met Val His His 1010 1015 1020Arg His
Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp Leu Thr 1025 1030
1035Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro Leu
1040 1045 1050Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly
Asp Leu 1055 1060 1065Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu
Pro Thr His Asp 1070 1075 1080Pro Ser Pro Leu Gln Arg Tyr Ser Glu
Asp Pro Thr Val Pro Leu 1085 1090 1095Pro Ser Glu Thr Asp Gly Tyr
Val Ala Pro Leu Thr Cys Ser Pro 1100 1105 1110Gln Pro Glu Tyr Val
Asn Gln Pro Asp Val Arg Pro Gln Pro Pro 1115 1120 1125Ser Pro Arg
Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala 1130 1135 1140Thr
Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val 1145 1150
1155Val Lys Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu
1160 1165 1170Tyr Leu Thr Pro Gln Gly Gly Ala Ala Pro Gln Pro His
Pro Pro 1175 1180 1185Pro Ala Phe Ser Pro Ala Phe Asp Asn Leu Tyr
Tyr Trp Asp Gln 1190 1195 1200Asp Pro Pro Glu Arg Gly Ala Pro Pro
Ser Thr Phe Lys Gly Thr 1205 1210 1215Pro Thr Ala Glu Asn Pro Glu
Tyr Leu Gly Leu Asp Val Pro Val 1220 1225 123010630PRTHomo Sapiens
10Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser1
5 10 15Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys
Gln 20 25 30Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn
Ala Ser 35 40 45Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr
Val Leu Ile 50 55 60Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg
Leu Arg Ile Val65 70 75 80Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr
Ala Leu Ala Val Leu Asp 85 90 95Asn Gly Asp Pro Leu Asn Asn Thr Thr
Pro Val Thr Gly Ala Ser Pro 100 105 110Gly Gly Leu Arg Glu Leu Gln
Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125Gly Gly Val Leu Ile
Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140Ile Leu Trp
Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr145 150 155
160Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met
165 170 175Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys
Gln Ser 180 185 190Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg
Cys Lys Gly Pro 195 200 205Leu Pro Thr Asp Cys Cys His Glu Gln Cys
Ala Ala Gly Cys Thr Gly 210 215 220Pro Lys His Ser Asp Cys Leu Ala
Cys Leu His Phe Asn His Ser Gly225 230 235 240Ile Cys Glu Leu His
Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255Phe Glu Ser
Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270Cys
Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280
285Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp
290 295 300Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg
Val Cys305 310 315 320Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val
Arg Ala Val Thr Ser 325 330 335Ala Asn Ile Gln Glu Phe Ala Gly Cys
Lys Lys Ile Phe Gly Ser Leu 340 345 350Ala Phe Leu Pro Glu Ser Phe
Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365Pro Leu Gln Pro Glu
Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380Thr Gly Tyr
Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu385 390 395
400Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn
405 410 415Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp
Leu Gly 420 425 430Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala
Leu Ile His His 435 440 445Asn Thr His Leu Cys Phe Val
His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460Arg Asn Pro His Gln
Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp465 470 475 480Glu Cys
Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490
495His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe
500 505 510Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln
Gly Leu 515 520 525Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro
Cys His Pro Glu 530 535 540Cys Gln Pro Gln Asn Gly Ser Val Thr Cys
Phe Gly Pro Glu Ala Asp545 550 555 560Gln Cys Val Ala Cys Ala His
Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575Arg Cys Pro Ser Gly
Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590Lys Phe Pro
Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605Thr
His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615
620Arg Ala Ser Pro Leu Thr625 63011231PRTHomo Sapiens 11Gly Pro Gly
Ser Ser Gly Lys Lys Pro Glu Ser Ala Ala Gly Ser Gln1 5 10 15Ser Pro
Ala Leu Pro Pro Gln Leu Lys Glu Met Lys Ser Gln Glu Ser 20 25 30Ala
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr 35 40
45Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg
50 55 60Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys
Ser65 70 75 80Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly
Glu Tyr Met 85 90 95Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala
Ser Ala Asn Ile 100 105 110Thr Ile Val Glu Ser Asn Glu Ile Ile Thr
Gly Met Pro Ala Ser Thr 115 120 125Glu Gly Ala Tyr Val Ser Ser Glu
Ser Pro Ile Arg Ile Ser Val Ser 130 135 140Thr Glu Gly Ala Asn Thr
Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly145 150 155 160Thr Ser His
Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val 165 170 175Asn
Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 180 185
190Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn
195 200 205Tyr Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe
Met Glu 210 215 220Ala Glu Glu Leu Tyr Gln Lys225 2301265PRTHomo
Sapiens 12Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro
Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg
Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile
Glu Phe Met Glu Ala 50 55 60Glu6513133PRTArtificialTtSlyD-FKBP-Her3
13Met Arg Ser Lys Val Gly Gln Asp Lys Val Val Thr Ile Arg Tyr Thr1
5 10 15Leu Gln Val Glu Gly Glu Val Leu Asp Gln Gly Glu Leu Ser Tyr
Leu 20 25 30His Gly His Arg Asn Leu Ile Pro Gly Leu Glu Glu Ala Leu
Glu Gly 35 40 45Arg Glu Glu Gly Glu Ala Phe Gln Ala His Val Pro Ala
Glu Lys Ala 50 55 60Tyr Gly Ala Gly Ser Pro Gln Pro Leu Val Tyr Asn
Lys Leu Thr Phe65 70 75 80Gln Leu Glu Pro Asn Pro His Thr Lys Gly
Ser Ser Gly Lys Asp Leu 85 90 95Asp Phe Gln Val Glu Val Val Lys Val
Arg Glu Ala Thr Pro Glu Glu 100 105 110Leu Leu His Gly His Ala His
Gly Gly Gly Ser Arg Lys His His His 115 120 125His His His His His
13014166PRTThermus thermophilus 14Met Arg Gly Ser Lys Val Gly Gln
Asp Lys Val Val Thr Ile Arg Tyr1 5 10 15Thr Leu Gln Val Glu Gly Glu
Val Leu Asp Gln Gly Glu Leu Ser Tyr 20 25 30Leu His Gly His Arg Asn
Leu Ile Pro Gly Leu Glu Glu Ala Leu Glu 35 40 45Gly Arg Glu Glu Gly
Glu Ala Phe Gln Ala His Val Pro Ala Glu Lys 50 55 60Ala Tyr Gly Pro
His Asp Pro Glu Gly Val Gln Val Val Pro Leu Ser65 70 75 80Ala Phe
Pro Glu Asp Ala Glu Val Val Pro Gly Ala Gln Phe Tyr Ala 85 90 95Gln
Asp Met Glu Gly Asn Pro Met Pro Leu Thr Val Val Ala Val Glu 100 105
110Gly Glu Glu Val Thr Val Asp Phe Asn His Pro Leu Ala Gly Lys Asp
115 120 125Leu Asp Phe Gln Val Glu Val Val Lys Val Arg Glu Ala Thr
Pro Glu 130 135 140Glu Leu Leu His Gly His Ala His Gly Gly Gly Ser
Arg Lys His His145 150 155 160His His His His His His
16515113PRTArtificialTtSlyDcas 15Met Arg Ser Lys Val Gly Gln Asp
Lys Val Val Thr Ile Arg Tyr Thr1 5 10 15Leu Gln Val Glu Gly Glu Val
Leu Asp Gln Gly Glu Leu Ser Tyr Leu 20 25 30His Gly His Arg Asn Leu
Ile Pro Gly Leu Glu Glu Ala Leu Glu Gly 35 40 45Arg Glu Glu Gly Glu
Ala Phe Gln Ala His Val Pro Ala Glu Lys Ala 50 55 60Tyr Gly Ala Gly
Ser Gly Ser Ser Gly Lys Asp Leu Asp Phe Gln Val65 70 75 80Glu Val
Val Lys Val Arg Glu Ala Thr Pro Glu Glu Leu Leu His Gly 85 90 95His
Ala His Gly Gly Gly Ser Arg Lys His His His His His His His 100 105
110His16128PRTArtificialTgSlyDdeltaIF 16Met Lys Val Glu Arg Gly Asp
Phe Val Leu Phe Asn Tyr Val Gly Arg1 5 10 15Tyr Glu Asn Gly Glu Val
Phe Asp Thr Ser Tyr Glu Ser Val Ala Arg 20 25 30Glu Gln Gly Ile Phe
Val Glu Glu Arg Glu Tyr Ser Pro Ile Gly Val 35 40 45Thr Val Gly Ala
Gly Glu Ile Ile Pro Gly Ile Glu Glu Ala Leu Leu 50 55 60Gly Met Glu
Leu Gly Glu Lys Lys Glu Val Val Val Pro Pro Glu Lys65 70 75 80Gly
Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr 85 90
95Ala Ile Phe Glu Ile Glu Val Val Glu Ile Lys Lys Ala Gly Glu Ala
100 105 110Leu Glu His His His His His His Leu Glu His His His His
His His 115 120 12517133PRTArtificialTtSlyDcas-Her3 17Met Arg Ser
Lys Val Gly Gln Asp Lys Val Val Thr Ile Arg Tyr Thr1 5 10 15Leu Gln
Val Glu Gly Glu Val Leu Asp Gln Gly Glu Leu Ser Tyr Leu 20 25 30His
Gly His Arg Asn Leu Ile Pro Gly Leu Glu Glu Ala Leu Glu Gly 35 40
45Arg Glu Glu Gly Glu Ala Phe Gln Ala His Val Pro Ala Glu Lys Ala
50 55 60Tyr Gly Ala Gly Ser Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr
Phe65 70 75 80Gln Leu Glu Pro Asn Pro His Thr Lys Gly Ser Ser Gly
Lys Asp Leu 85 90 95Asp Phe Gln Val Glu Val Val Lys Val Arg Glu Ala
Thr Pro Glu Glu 100 105 110Leu Leu His Gly His Ala His Gly Gly Gly
Ser Arg Lys His His His 115 120 125His His His His His
13018130PRTArtificialTtSlyDcys-Her3 18Met Arg Gly Ser Lys Val Gly
Gln Asp Lys Val Val Thr Ile Arg Tyr1 5 10 15Thr Leu Gln Val Glu Gly
Glu Val Leu Asp Gln Gly Glu Leu Ser Tyr 20 25 30Leu His Gly His Arg
Asn Leu Ile Pro Gly Leu Glu Glu Ala Leu Glu 35 40 45Gly Arg Glu Glu
Gly Glu Ala Phe Gln Ala His Val Pro Ala Glu Lys 50 55 60Ala Tyr Gly
Pro Cys Gly Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr65 70 75 80Phe
Gln Leu Glu Pro Asn Pro His Thr Gly Cys Gly Lys Asp Leu Asp 85 90
95Phe Gln Val Glu Val Val Lys Val Arg Glu Ala Thr Pro Glu Glu Leu
100 105 110Leu His Gly His Ala His Gly Gly Gly Ser His His His His
His His 115 120 125His His 13019142PRTArtificialTgSlyDser-Her3
19Met Lys Val Glu Arg Gly Asp Phe Val Leu Phe Asn Tyr Val Gly Arg1
5 10 15Tyr Glu Asn Gly Glu Val Phe Asp Thr Ser Tyr Glu Ser Val Ala
Arg 20 25 30Glu Gln Gly Ile Phe Val Glu Glu Arg Glu Tyr Ser Pro Ile
Gly Val 35 40 45Thr Val Gly Ala Gly Glu Ile Ile Pro Gly Ile Glu Glu
Ala Leu Leu 50 55 60Gly Met Glu Leu Gly Glu Lys Lys Glu Val Val Val
Pro Pro Glu Lys65 70 75 80Gly Tyr Gly Met Pro Ser Gly Pro Gln Pro
Leu Val Tyr Asn Lys Leu 85 90 95Thr Phe Gln Leu Glu Pro Asn Pro His
Thr Gly Ser Ala Gly Lys Thr 100 105 110Ala Ile Phe Glu Ile Glu Val
Val Glu Ile Lys Lys Ala Gly Glu Ala 115 120 125Gly Gly Gly Ser Arg
Lys His His His His His His His His 130 135
14020143PRTArtificialTgSlyDcys-Her3 20Met Arg Gly Ser Lys Val Glu
Arg Gly Asp Phe Val Leu Phe Asn Tyr1 5 10 15Val Gly Arg Tyr Glu Asn
Gly Glu Val Phe Asp Thr Ser Tyr Glu Ser 20 25 30Val Ala Arg Glu Gln
Gly Ile Phe Val Glu Glu Arg Glu Tyr Ser Pro 35 40 45Ile Gly Val Thr
Val Gly Ala Gly Glu Ile Ile Pro Gly Ile Glu Glu 50 55 60Ala Leu Leu
Gly Met Glu Leu Gly Glu Lys Lys Glu Val Val Val Pro65 70 75 80Pro
Glu Lys Gly Tyr Gly Met Pro Cys Gly Pro Gln Pro Leu Val Tyr 85 90
95Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn Pro His Thr Gly Cys Ala
100 105 110Gly Lys Thr Ala Ile Phe Glu Ile Glu Val Val Glu Ile Lys
Lys Ala 115 120 125Gly Glu Ala Gly Gly Gly Ser His His His His His
His His His 130 135 14021133PRTArtificialTtSlyDcas-Her4 21Met Arg
Ser Lys Val Gly Gln Asp Lys Val Val Thr Ile Arg Tyr Thr1 5 10 15Leu
Gln Val Glu Gly Glu Val Leu Asp Gln Gly Glu Leu Ser Tyr Leu 20 25
30His Gly His Arg Asn Leu Ile Pro Gly Leu Glu Glu Ala Leu Glu Gly
35 40 45Arg Glu Glu Gly Glu Ala Phe Gln Ala His Val Pro Ala Glu Lys
Ala 50 55 60Tyr Gly Ala Gly Ser Pro Gln Thr Phe Val Tyr Asn Pro Thr
Thr Phe65 70 75 80Gln Leu Glu His Asn Phe Asn Ala Lys Gly Ser Ser
Gly Lys Asp Leu 85 90 95Asp Phe Gln Val Glu Val Val Lys Val Arg Glu
Ala Thr Pro Glu Glu 100 105 110Leu Leu His Gly His Ala His Gly Gly
Gly Ser Arg Lys His His His 115 120 125His His His His His
13022130PRTArtificialTtSlyDcys-Her4 22Met Arg Gly Ser Lys Val Gly
Gln Asp Lys Val Val Thr Ile Arg Tyr1 5 10 15Thr Leu Gln Val Glu Gly
Glu Val Leu Asp Gln Gly Glu Leu Ser Tyr 20 25 30Leu His Gly His Arg
Asn Leu Ile Pro Gly Leu Glu Glu Ala Leu Glu 35 40 45Gly Arg Glu Glu
Gly Glu Ala Phe Gln Ala His Val Pro Ala Glu Lys 50 55 60Ala Tyr Gly
Pro Cys Gly Pro Gln Thr Phe Val Tyr Asn Pro Thr Thr65 70 75 80Phe
Gln Leu Glu His Asn Phe Asn Ala Gly Cys Gly Lys Asp Leu Asp 85 90
95Phe Gln Val Glu Val Val Lys Val Arg Glu Ala Thr Pro Glu Glu Leu
100 105 110Leu His Gly His Ala His Gly Gly Gly Ser His His His His
His His 115 120 125His His 13023142PRTArtificialTgSlyDser-Her4
23Met Lys Val Glu Arg Gly Asp Phe Val Leu Phe Asn Tyr Val Gly Arg1
5 10 15Tyr Glu Asn Gly Glu Val Phe Asp Thr Ser Tyr Glu Ser Val Ala
Arg 20 25 30Glu Gln Gly Ile Phe Val Glu Glu Arg Glu Tyr Ser Pro Ile
Gly Val 35 40 45Thr Val Gly Ala Gly Glu Ile Ile Pro Gly Ile Glu Glu
Ala Leu Leu 50 55 60Gly Met Glu Leu Gly Glu Lys Lys Glu Val Val Val
Pro Pro Glu Lys65 70 75 80Gly Tyr Gly Met Pro Ser Gly Pro Gln Thr
Phe Val Tyr Asn Pro Thr 85 90 95Thr Phe Gln Leu Glu His Asn Phe Asn
Ala Gly Ser Ala Gly Lys Thr 100 105 110Ala Ile Phe Glu Ile Glu Val
Val Glu Ile Lys Lys Ala Gly Glu Ala 115 120 125Gly Gly Gly Ser Arg
Lys His His His His His His His His 130 135
14024143PRTArtificialTgSlyDcys-Her4 24Met Arg Gly Ser Lys Val Glu
Arg Gly Asp Phe Val Leu Phe Asn Tyr1 5 10 15Val Gly Arg Tyr Glu Asn
Gly Glu Val Phe Asp Thr Ser Tyr Glu Ser 20 25 30Val Ala Arg Glu Gln
Gly Ile Phe Val Glu Glu Arg Glu Tyr Ser Pro 35 40 45Ile Gly Val Thr
Val Gly Ala Gly Glu Ile Ile Pro Gly Ile Glu Glu 50 55 60Ala Leu Leu
Gly Met Glu Leu Gly Glu Lys Lys Glu Val Val Val Pro65 70 75 80Pro
Glu Lys Gly Tyr Gly Met Pro Cys Gly Pro Gln Thr Phe Val Tyr 85 90
95Asn Pro Thr Thr Phe Gln Leu Glu His Asn Phe Asn Ala Gly Cys Ala
100 105 110Gly Lys Thr Ala Ile Phe Glu Ile Glu Val Val Glu Ile Lys
Lys Ala 115 120 125Gly Glu Ala Gly Gly Gly Ser His His His His His
His His His 130 135 140256PRTMus musculus 25Asp Tyr Trp Ile His
Trp1 52616PRTMus musculus 26Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser
Asn Gln Lys Phe Lys Asp1 5 10 15277PRTMus musculus 27Pro Tyr Tyr
Tyr Gly Asp Tyr1 52810PRTMus musculus 28Ser Ala Ser Ser Ser Val Ser
Tyr Met His1 5 10297PRTMus musculus 29Ser Thr Ser Asn Leu Ala Ser1
5309PRTMus musculus 30Gln Gln Arg Ser Ser Tyr Pro Phe Thr1
531116PRTMus musculus 31Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Ala Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Thr Ser Gly
Tyr Thr Leu Thr Asp Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly
Tyr Thr Glu Ser Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Ile Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr
Tyr Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val
Ser Ser 11532106PRTMus musculus 32Gln Ile Val Leu Thr Gln Ser Pro
Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Phe Gln Gln Lys
Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser Asn Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90 95Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys 100
10533116PRTArtificialhumanized variant A of heavy chain
variable
domain VH of M-05-74 (VH-A) 33Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Arg Thr
Ser Gly Tyr Thr Leu Thr Asp Tyr 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr
Thr Gly Tyr Thr Glu Ser Asn Gln Lys Phe 50 55 60Lys Asp Arg Val Ala
Met Thr Arg Asp Ala Ser Ile Asn Thr Ala Tyr65 70 75 80Met Glu Leu
Thr Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Pro Tyr Tyr Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 11534116PRTArtificialhumanized variant B of
heavy chain variable domain VH of M-05-74 (VH-B) 34Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp Tyr 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly
Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser Asn Gln Lys Phe 50 55
60Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Asn Thr Ala Tyr65
70 75 80Met Ala Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly Asp Tyr Trp Gly Gln Gly Thr
Leu Val 100 105 110Thr Val Ser Ser 11535116PRTArtificialhumanized
variant C of heavy chain variable domain VH of M-05-74 (VH-C) 35Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Ser Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr
20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser Asn Gln
Lys Phe 50 55 60Lys Asp Arg Val Thr Leu Ile Arg Asp Thr Ser Thr Thr
Thr Val Tyr65 70 75 80Met Glu Leu Thr Ser Leu Thr Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly Asp Tyr Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11536116PRTArtificialhumanized variant D of heavy chain variable
domain VH of M-05-74 (VH-D) 36Gln Val Gln Leu Val Gln Ser Gly Gly
Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Leu Thr Asp Tyr 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr
Thr Gly Tyr Thr Glu Ser Asn Gln Lys Phe 50 55 60Lys Asp Arg Val Thr
Met Thr Ala Asp Ala Ser Thr Gly Thr Ala Tyr65 70 75 80Ile Glu Leu
Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Pro Tyr Tyr Tyr Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 11537116PRTArtificialhumanized variant E of
heavy chain variable domain VH of M-05-74 (VH-E) 37Gln Val Gln Leu
Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp Tyr 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser Asn Gln Lys Phe 50 55
60Lys Asp Arg Val Thr Met Thr Ser Asp Thr Ser Ile Asp Thr Ala Tyr65
70 75 80Met Glu Leu Thr Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly Asp Tyr Trp Gly Gln Gly Thr
Thr Val 100 105 110Thr Val Ser Ser 11538106PRTArtificialhumanized
variant A of light chain variable domain VL of M-05-74 (VL-A) 38Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr 35 40 45Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser
Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Ser Tyr Pro Phe Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 10539106PRTArtificialhumanized variant B of light chain
variable domain VL of M-05-74 (VL-B) 39Glu Ile Val Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser Asn
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100
10540106PRTArtificialhumanized variant C of light chain variable
domain VL of M-05-74 (VL-C) 40Glu Ile Val Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Phe Gln Gln Lys Pro
Gly Lys Ala Pro Lys Ser Leu Ile Tyr 35 40 45Ser Thr Ser Asn Leu Ala
Ser Gly Val Pro Ser Lys Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90 95Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 10541106PRTArtificialhumanized
variant D of light chain variable domain VL of M-05-74 (VL-D) 41Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30His Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile
Tyr 35 40 45Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser
Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Ser Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 10542106PRTArtificialhumanized variant E of light chain
variable domain VL of M-05-74 (VL-E) 42Glu Ile Val Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser Asn
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90
95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 1054311PRTHomo
Sapiens 43Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro1 5
104410PRTHomo Sapiens 44Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu1 5
1045234PRTArtificialPseudomonas exotoxin variant PE24LR8M_3G
(including a GGG linker) 45Met Gly Gly Gly Arg His Arg Gln Pro Arg
Gly Trp Glu Gln Leu Tyr1 5 10 15Pro Thr Gly Ala Glu Phe Leu Gly Asp
Gly Gly Ala Val Ser Phe Ser 20 25 30Thr Arg Gly Thr Gln Asn Trp Thr
Val Glu Arg Leu Leu Gln Ala His 35 40 45Arg Gln Leu Glu Glu Gly Gly
Tyr Val Phe Val Gly Tyr His Gly Thr 50 55 60Phe Leu Glu Ala Ala Gln
Ser Ile Val Phe Gly Gly Val Arg Ala Arg65 70 75 80Ser Gln Asp Leu
Asp Ala Ile Trp Ala Gly Phe Tyr Ile Ala Gly Asp 85 90 95Pro Ala Leu
Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Ala 100 105 110Gly
Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser 115 120
125Ser Leu Pro Gly Phe Tyr Ala Thr Ser Leu Thr Leu Ala Ala Pro Glu
130 135 140Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro
Leu Arg145 150 155 160Leu Asp Ala Ile Thr Gly Pro Glu Glu Ser Gly
Gly Arg Leu Glu Thr 165 170 175Ile Leu Gly Trp Pro Leu Ala Glu Arg
Thr Val Val Ile Pro Ser Ala 180 185 190Ile Pro Thr Asp Pro Arg Asn
Val Gly Gly Asp Leu Asp Pro Ser Ser 195 200 205Ile Pro Asp Ser Glu
Ala Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser 210 215 220Gln Pro Gly
Lys Pro Pro Arg Glu Asp Leu225 23046213PRTArtificialLight chain of
M-05-74 (M-05-74_LC) 46Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Ser Ala Ser
Ser Ser Val Ser Tyr Met 20 25 30His Trp Phe Gln Gln Lys Pro Gly Thr
Ser Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser Asn Leu Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90 95Phe Gly Ser Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120
125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
21047238PRTArtificialHeavy chain of M-05-74 HC with sortase tag
(M-05-74_HC) 47Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys
Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr
Leu Thr Asp Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr
Glu Ser Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Ile Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr
Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135
140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Gly Gly Gly Ser Leu 210 215 220Pro Glu Thr Gly
Gly Ser Gly Ser His His His His His His225 230
23548460PRTArtificialHeavy chain of M-05-74 HC conjugated to
Pseudomonas exotoxin variant PE24LR8M (Fab-074-PE heavy chain 1)
48Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp
Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser Asn
Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Ala Tyr65 70 75 80Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly Asp Tyr
Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155
160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Gly Gly Gly Ser Leu 210 215 220Pro Glu Thr Gly Gly Gly Arg His
Arg Gln Pro Arg Gly Trp Glu Gln225 230 235 240Leu Tyr Pro Thr Gly
Ala Glu Phe Leu Gly Asp Gly Gly Ala Val Ser 245 250 255Phe Ser Thr
Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln 260 265 270Ala
His Arg Gln Leu Glu Glu Gly Gly Tyr Val Phe Val Gly Tyr His 275 280
285Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg
290 295 300Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Ala Gly Phe Tyr
Ile Ala305 310 315 320Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln
Asp Gln Glu Pro Asp 325 330 335Ala Ala Gly Arg Ile Arg Asn Gly Ala
Leu Leu Arg Val Tyr Val Pro 340 345 350Arg Ser Ser Leu Pro Gly Phe
Tyr Ala Thr Ser Leu Thr Leu Ala Ala 355 360 365Pro Glu Ala Ala Gly
Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro 370 375 380Leu Arg Leu
Asp Ala Ile Thr Gly Pro Glu Glu Ser Gly Gly Arg Leu385 390 395
400Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro
405 410 415Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu
Asp Pro 420 425 430Ser Ser Ile Pro Asp Ser Glu Ala Ala Ile Ser Ala
Leu Pro Asp Tyr 435 440 445Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu
Asp Leu 450 455 46049457PRTArtificialHeavy chain of M-05-74 HC
conjugated to Pseudomonas exotoxin variant PE24LR8M (Fab-074-PE
heavy chain 2) as direct PE24LR8M fusion 49Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala1 5
10 15Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp
Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu Ser Asn
Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Ala Tyr65 70 75 80Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly Asp Tyr
Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155
160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Lys Ala Ser Gly Gly 210 215 220Arg His Arg Gln Pro Arg Gly Trp
Glu Gln Leu Gly Gly Ser Pro Thr225 230 235 240Gly Ala Glu Phe Leu
Gly Asp Gly Gly Ala Val Ser Phe Ser Thr Arg 245 250 255Gly Thr Gln
Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln 260 265 270Leu
Glu Glu Gly Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu 275 280
285Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln
290 295 300Asp Leu Asp Ala Ile Trp Ala Gly Phe Tyr Ile Ala Gly Asp
Pro Ala305 310 315 320Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro
Asp Ala Ala Gly Arg 325 330 335Ile Arg Asn Gly Ala Leu Leu Arg Val
Tyr Val Pro Arg Ser Ser Leu 340 345 350Pro Gly Phe Tyr Ala Thr Ser
Leu Thr Leu Ala Ala Pro Glu Ala Ala 355 360 365Gly Glu Val Glu Arg
Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp 370 375 380Ala Ile Thr
Gly Pro Glu Glu Ser Gly Gly Arg Leu Glu Thr Ile Leu385 390 395
400Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro
405 410 415Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser
Ile Pro 420 425 430Asp Ser Glu Ala Ala Ile Ser Ala Leu Pro Asp Tyr
Ala Ser Gln Pro 435 440 445Gly Lys Pro Pro Arg Glu Asp Leu Lys 450
45550147PRTStaphylococcus aureus 50Gln Ala Lys Pro Gln Ile Pro Lys
Asp Lys Ser Lys Val Ala Gly Tyr1 5 10 15Ile Glu Ile Pro Asp Ala Asp
Ile Lys Glu Pro Val Tyr Pro Gly Pro 20 25 30Ala Thr Pro Glu Gln Leu
Asn Arg Gly Val Ser Phe Ala Glu Glu Asn 35 40 45Glu Ser Leu Asp Asp
Gln Asn Ile Ser Ile Ala Gly His Thr Phe Ile 50 55 60Asp Arg Pro Asn
Tyr Gln Phe Thr Asn Leu Lys Ala Ala Lys Lys Gly65 70 75 80Ser Met
Val Tyr Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr Lys Met 85 90 95Thr
Ser Ile Arg Asp Val Lys Pro Thr Asp Val Gly Val Leu Asp Glu 100 105
110Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp Asp Tyr
115 120 125Asn Glu Lys Thr Gly Val Trp Glu Lys Arg Lys Ile Phe Val
Ala Thr 130 135 140Glu Val Lys14551119PRTMus musculus 51Gln Ala Tyr
Leu Gln Gln Ser Gly Ala Glu Leu Met Arg Pro Gly Ala1 5 10 15Ser Val
Arg Met Ser Cys Gln Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr
Ile His Trp Leu Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile 35 40
45Gly Ala Ile Tyr Pro Arg Asn Gly Asp Phe Ser Tyr Asn Gln Lys Phe
50 55 60Lys Asp Lys Ala Ser Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr65 70 75 80Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95Ala Arg Thr Ile Asn Tyr Gly Asp Trp Phe Ala Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala
11552107PRTMus musculus 52Asp Ile Gln Met Ile Gln Ser Pro Ala Ser
Leu Phe Val Ser Glu Gly1 5 10 15Glu Thr Val Ile Ile Thr Cys Arg Ala
Ser Glu Asn Ile Tyr Ser Asn 20 25 30Leu Ala Trp Tyr His Gln Lys Lys
Gly Lys Ser Pro Gln Val Leu Val 35 40 45Tyr Ala Ala Ile Lys Leu Ala
Asp Gly Val Pro Leu Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln
Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser65 70 75 80Glu Asp Phe Gly
Ser Tyr Tyr Cys Gln His Phe Trp Gly Pro Pro Tyr 85 90 95Thr Phe Gly
Ser Gly Thr Asn Leu Glu Ile Lys 100 10553107PRTHomo Sapiens 53Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10554105PRTHomo Sapiens 54Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys
Thr Val Ala Pro Thr Glu Cys Ser 100 10555330PRTHomo Sapiens 55Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33056330PRThomo sapiens 56Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 33057330PRThomo sapiens 57Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33058327PRTHomo
Sapiens 58Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150
155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys
32559157PRTHomo sapiens 59Arg Ser Arg Ala Cys His Pro Cys Ser Pro
Met Cys Lys Gly Ser Arg1 5 10 15Cys Trp Gly Glu Ser Ser Glu Asp Cys
Gln Ser Leu Thr Arg Thr Val 20 25 30Cys Ala Gly Gly Cys Ala Arg Cys
Lys Gly Pro Leu Pro Thr Asp Cys 35 40 45Cys His Glu Gln Cys Ala Ala
Gly Cys Thr Gly Pro Lys His Ser Asp 50 55 60Cys Leu Ala Cys Leu His
Phe Asn His Ser Gly Ile Cys Glu Leu His65 70 75 80Cys Pro Ala Leu
Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro 85 90 95Asn Pro Glu
Gly Arg Tyr Thr Phe Gly Ala Ser
Cys Val Thr Ala Cys 100 105 110Pro Tyr Asn Tyr Leu Ser Thr Asp Val
Gly Ser Cys Thr Leu Val Cys 115 120 125Pro Leu His Asn Gln Glu Val
Thr Ala Glu Asp Gly Thr Gln Arg Cys 130 135 140Glu Lys Cys Ser Lys
Pro Cys Ala Arg Val Cys Tyr Gly145 150 155605PRTArtificialheavy
chain HVR-H1, pertuzumab 60Asp Tyr Thr Met Asp1
56117PRTArtificialheavy chain HVR-H2 pertuzumab 61Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys1 5 10
15Gly6210PRTArtificialheavy chain HVR-H3, pertuzumab 62Asn Leu Gly
Pro Ser Phe Tyr Phe Asp Tyr1 5 106311PRTArtificiallight chain
HVR-L1, pertuzumab 63Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala1 5
10647PRTArtificiallight chain HVR-L2, pertuzumab 64Ser Ala Ser Tyr
Arg Tyr Thr1 5659PRTArtificiallight chain HVR-L3 pertuzumab 65Gln
Gln Tyr Tyr Ile Tyr Pro Tyr Thr1 566119PRTArtificialheavy chain
variable domain VH, pertuzumab 66Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Thr Met Asp Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Phe
Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 11567107PRTArtificiallight chain
variable domain VL, pertuzumab 67Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10568453PRTArtificialheavy chain 1, bispecific HER3/HER2 antibody
DIBxPERT 68Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser
Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe 115 120 125Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 130 135 140Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp145 150
155 160Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr 165 170 175Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr 180 185 190Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala Cys Glu Val 195 200 205Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 210 215 220Glu Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235 240Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265
270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val Ser Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390
395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
45069212PRTArtificiallight chain 1, bispecific HER3/HER2 antibody
DIBxPERT 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val
Ser Ile Gly 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Ser Ser Ala Ser Thr 100 105 110Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 115 120 125Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 130 135 140Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His145 150
155 160Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser 165 170 175Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys 180 185 190Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu 195 200 205Pro Lys Ser Cys
21070446PRTArtificialheavy chain 2, bispecific HER3/HER2 antibody
DIBxPERT 70Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro
Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Leu
Thr Asp Tyr 20 25 30Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Gly Tyr Thr Glu
Ser Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys
Ser Ser Asn Thr Ala Tyr65 70 75 80Ile Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Tyr Tyr Tyr Gly
Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Cys Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 44571213PRTArtificiallight chain 2, bispecific
HER3/HER2 antibody DIBxPERT 71Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Phe Gln Gln Lys Pro
Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45Ser Thr Ser Asn Leu Ala
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe Thr 85 90 95Phe Gly
Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 210
* * * * *